Formation of layers of amphiphilic molecules

ABSTRACT

To form a layer separating two volumes of aqueous solution, there is used an apparatus comprising elements defining a chamber, the elements including a body of non-conductive material having formed therein at least one recess opening into the chamber, the recess containing an electrode. A pre-treatment coating of a hydrophobic fluid is applied to the body across the recess. Aqueous solution, having amphiphilic molecules added thereto, is flowed across the body to cover the recess so that aqueous solution is introduced into the recess from the chamber and a layer of the amphiphilic molecules forms across the recess separating a volume of aqueous solution introduced into the recess from the remaining volume of aqueous solution.

RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/302,287 filed Jun. 11, 2014, now abandoned, which is acontinuation of U.S. patent application Ser. No. 12/339,956 filed Dec.19, 2008, now abandoned, which claims foreign priority benefits under 35U.S.C. § 119(a)-(d) or 35 U.S.C. § 365(b) of United Kingdom PatentApplication No. 0724736.4 filed on Dec. 19, 2007, and which also claimspriority under 35 U.S.C. § 119(e) to U.S. Provisional Patent ApplicationSer. No. 61/080,492 filed Jul. 14, 2008. The entire contents of theabove referenced applications are incorporated herein by reference intheir entirety.

FIELD OF THE DISCLOSURE

In one aspect, the present disclosure relates to the formation of layersof amphiphilic molecules such as lipid bilayers. It is particularlyconcerned with the formation of high quality layers suitable forapplications requiring measurement of electrical signals with a highdegree of sensitivity, for example single channel recordings andstochastic sensing for biosensor or drug screening applications. In someaspects, it is concerned with applications employing arrays of layers ofamphiphilic molecules, for example lipid bilayers. In another aspect,the present disclosure relates to the performance of an electrodeprovided in a recess, for example for conducting electro-physiologicalmeasurements.

BACKGROUND OF THE DISCLOSURE

The potential for using cellular proteins for biosensing and drugdiscovery applications has long been appreciated. However there are manytechnical challenges to overcome in developing this technology to fullyrealise the potential. There is a wealth of literature on usingfluorescent and optical approaches, but the focus of this document is onthe measurement of electrical signals to recognise analytes inbiosensing.

In one type of technique, a layer of amphiphilic molecules may be usedas the layer separating two volumes of aqueous solution. The layerresists the flow of current between the volumes. A membrane protein isinserted into the layer to selectively allow the passage of ions acrossthe layer, which is recorded as an electrical signal detected byelectrodes in the two volumes of aqueous solution. The presence of atarget analyte modulates the flow of ions and is detected by observingthe resultant variations in the electrical signal. Such techniquestherefore allow the layer to be used as a biosensor to detect theanalyte. The layer is an essential component of the single moleculebiosensor presented and its purpose is two-fold. Firstly the layerprovides a platform for the protein which acts as a sensing element.Secondly the layer isolates the flow of ions between the volumes, theelectrical resistance of the layer ensuring that the dominantcontribution of ionic flow in the system is through the membrane proteinof interest, with negligible flow through the bilayer, thus allowingdetection of single protein channels.

A specific application is stochastic sensing, where the number ofmembrane proteins is kept small, typically between 1 and 100, so thatthe behaviour of a single protein molecule can be monitored. This methodgives information on each specific molecular interaction and hence givesricher information than a bulk measurement. However, due to the smallcurrents involved, typically a few pA, requirements of this approach area very high resistance seal, typically at least 1 GΩ and for someapplications one or two orders of magnitude higher, and sufficientelectrical sensitivity to measure the currents. While the requirementsfor stochastic sensing have been met in the laboratory, the conditionsand expertise required limit its use. In addition, the laboratorymethods are laborious and time-consuming and are not easily scalable tohigh-density arrays, which are desirable for any commercial biosensor.Furthermore, the fragility of single bilayer membranes means thatanti-vibration tables are often employed in the laboratory.

By way of background, existing techniques for forming layers ofamphiphilic molecules such as lipid bilayers will be reviewed.

Several methods for forming planar artificial lipid bilayers are knownin the art, most notably including folded bilayer formation (e.g. Montal& Mueller method), tip-dipping, painting, patch clamping, andwater-in-oil droplet interfaces.

At present, the bulk of routine single ion channel characterisation inresearch labs is performed using folded bilayers, painted bilayers ortip-dip methods. These methods are used either for the ease of bilayerformation, or for the high resistive seals that can be formed (e.g.10-100 GΩ). Tip-dip bilayers and bilayers from patch-clamping of giantunilamellar liposomes are also studied as they can be formed in asolvent free manner, which is thought to be important for the activityof some protein channels. The method of Montal & Mueller (Proc. Natl.Acad. Sci. USA. (1972), 69, 3561-3566) is popular as a cost-effectiveand relatively straightforward method of forming good quality foldedlipid bilayers suitable for protein pore insertion, in which a lipidmonolayer is carried on the water/air interface past either side of anaperture in a membrane which is perpendicular to that interface.Typically, the lipid is added to the surface of the aqueous electrolytesolution by first dissolving it in an organic solvent, a drop of whichis then allowed to evaporate on the surface of the aqueous solution oneither side of the aperture. Once the organic solvent has beenevaporated, the solution/air interfaces are physically moved up and downpast either side of the aperture until a bilayer is formed. Thetechnique requires the presence of a hydrophobic oil applied as apre-treatment coating to the aperture surface. The primary function ofthe hydrophobic oil is to form an annulus region between the bilayer andthe aperture film where the lipid monolayers must come together over adistance typically between 1 and 25 μm.

Tip-dipping bilayer formation entails touching the aperture surface(e.g. a pipette tip) onto the surface of a test solution that iscarrying a monolayer of lipid. Again the lipid monolayer is firstgenerated at the solution/air interface by evaporating a drop of lipiddissolved in organic solvent applied to the solution surface. Thebilayer is then formed by mechanical actuation to move the apertureinto/out of the solution surface.

For painted bilayers, the drop of lipid dissolved in organic solvent isapplied directly to the aperture, which is submerged in the aqueous testsolution. The lipid solution is spread thinly over the aperture using apaint brush or equivalent. Thinning of the solvent results in formationof a lipid bilayer, however, complete removal of the solvent from thebilayer is difficult and consequently the bilayer formed is less stableand more noise prone during measurement.

Patch-clamping is commonly used in the study of biological cellmembranes, whereby the cell membrane is clamped to the end of a pipetteby suction and a patch of the membrane becomes attached over theaperture. The method has been adapted for artificial bilayer studies byclamping liposomes which then burst to leave a lipid bilayer sealingover the aperture of the pipette. This requires stable giant unilamellarliposomes and the fabrication of small apertures in glass surfacedmaterials.

Water-in-oil droplet interfaces are a more recent disclosure in whichtwo aqueous samples are submerged in a reservoir of hydrocarbon oilcontaining lipid. The lipid accumulates in a monolayer at the oil/waterinterface such that when the two samples are brought into contact abilayer is formed at the interface between them.

In any of these techniques, once the bilayer has been formed, theprotein is then introduced to the bilayer either by random collisionfrom the aqueous solution, by fusion of vehicles containing the protein,or by mechanically transporting it to the bilayer, for example on theend of a probe device such as an agar tipped rod.

There have been great efforts recently to increase the ease of bilayerformation using micro fabrication. Some techniques have attemptedessentially to miniaturise standard systems for folded lipid bilayers.Other techniques include bilayer formation on solid substrates ordirectly on electrode surfaces, through either covalent attachment orphysical adsorption.

A large proportion of the devices that are capable of performingstochastic sensing form a bilayer by using a variant of the folded lipidbilayers technique or the painted bilayer technique. To date most haveconcentrated either on novel methods of aperture formation or onutilising the emerging technologies in micro fabrication to miniaturisethe device or to create a plurality of addressable sensors.

An example is Suzuki et al., “Planar lipid bilayer reconstitution with amicro-fluidic system”, Lab Chip, (4), 502-505, 2004. Herein, an aperturearray is created by etching a silicon substrate, followed by a surfacetreatment to encourage the bilayer formation process, although thedisclosed rate of successful bilayer formation is very low (two out often).

A more recent example is disclosed in Sandison, et al., “Air exposuretechnique for the formation of artificial lipid bilayers inmicrosystems”, Langmuir, (23), 8277-8284, 2007. Herein the devicefabricated from poly(methylmethacrylate) contains two distinct aqueouschambers. Problems with the reproducibility of bilayer formation areattributed to the difficulty in removing the excess hydrophobic materialfrom the aperture, and tackled by using a period of air exposure to aidthe bilayer formation process to thin the pre-treatment.

The devices of both Sandison et al. and Suzuki et al. are bothminiaturised versions of a standard painted bilayer technique with twodistinct fluidic chambers separated by a septum containing an apertureacross which the bilayer is formed, one chamber being filled before theother. This presents a number of difficulties for scaling up the systemto a large number of individually addressable bilayers, as at least oneof the aqueous chambers must be a distinct chamber with no electrical orionic connectivity to any other chamber. Sandison et al. created adevice with three fluid chambers, each with separate fluidics, anapproach which would be difficult to scale to large numbers of bilayers.Suzuki et al. tried to address this problem by using a hydrophobicphotoresist layer to create small aqueous chambers on top of theaperture containing substrate. In this case, it is difficult to controlthe flow of solution across the aperture containing interface and theuse of small volumes exposed to air makes the apparatus susceptible toevaporation effects. In both cited examples, the need for the individualaqueous chambers for each bilayer means that a large sample volume mustbe used to fill all the chambers.

An example of biosensor device using a supported lipid bilayer isdisclosed in U.S. Pat. No. 5,234,566. The device is capacitive. A gatedion channel responds to an analyte, the binding of this analyte causes achange in the gating behavior of the ion channel, and this is measuredvia the electrical response of the membrane capacitance. To support thelipid bilayer, there is used a monolayer of alkane-thiol molecules on agold electrode, which provides a scaffold for a lipid monolayer toself-assemble onto. This monolayer can incorporate ion channels such asgramicidin which are used as the sensing element of the device.Variations on this method have been used to create a tethered lipidbilayer onto an electrode surface to incorporate other membraneproteins. However, the approach has a number of drawbacks, the first isthat the small aqueous volume present under the lipid bilayer, typicallyof the order of 1 nm to 10 nm thick, does not contain enough ions toperform a direct current measurement for any useful period of time. Thisis an effect common to nearly all tethered bilayer systems on solidsupports. For recordings of any meaningful duration, an alternatingcurrent measurement must be used to overcome the ionic depletion at theelectrode, but that limits the sensitivity of the device.

An example of a biosensor device using a supported lipid bilayer isdisclosed in Urisu et al., “Formation of high-resistance supported lipidbilayer on the surface of a silicon substrate with micro electrodes”,Nanomedicine, 2005, (1), 317-322. This device exploits the strongsurface adhesion between phospholipid molecules and a SiO₂ surface toform a supported bilayer. A silicon oxide surface is modified, usingetching techniques common in silicon chip production, to expose smallchannels to an electrode surface. A bilayer is then formed on thesilicon oxide surface, resulting in an electrical resistance of a fewMΩ. In this system, the wells created by this process could not beindividually addressed.

In both of the cited examples using a supported lipid bilayer, it isvery difficult to form a high resistive seal using these methods.Although the resistance may be sufficient to observe a change arisingfrom a large number of ion channels, single channel or stochasticmeasurements, which are inherently more sensitive, are incrediblychallenging using this methodology.

There are a number of problems with the supported bilayer approach inthese documents and in general, which makes this system unsuitable. Thefirst problem lies with the resistance of the bilayer membrane which istypically about 100MΩ. While this may be suitable for examining proteinbehaviour at large protein concentrations, it is not sufficient for ahigh-fidelity assay based on single molecule sensing, typicallyrequiring a resistance of at least 1 GΩ and for some applications one ortwo orders of magnitude higher. The second problem is the small volumeof solution trapped in the short distance between the bilayer and thesolid support, typically of the order of 1 nm. This small volume doesnot contain many ions, affecting the stability of the potential acrossthe bilayer and limiting the duration of the recording.

A number of methods have been proposed to overcome the problems withsolid supported bilayers. One option is to incorporate a chemicallinkage between the bilayer and the surface, either a small polyethyleneglycol layer is introduced (polymer cushioned bilayers), or the lipid ischemically modified to contain a small hydrophilic linkage and reactedwith the surface providing a scaffold for vehicle deposition (tetheredbilayers). While these methods have increased the ionic reservoirbeneath the lipid bilayer, they are inconvenient to implement and havedone little to decrease the current leakage across the bilayer.

The techniques used in the silicon chip industry provide an attractivetechnology for creating a large number of electrodes that could be usedin biosensor applications. This approach is disclosed in the relatedapplications U.S. Pat. Nos. 7,144,486 and 7,169,272. U.S. Pat. No.7,144,486 discloses a method of fabricating a microelectrode devicecontaining microcavities etched into layers of insulator material. Thedevices are said to have a wide range of electrochemical applications inwhich electrodes in the cavities measure electrical signals. It isstated that thin films may be suspended across the cavities. Severaltypes of film are mentioned, including being a lipid bilayer. Howeverthis is merely a proposal and there is no disclosure of any techniquefor forming the lipid bilayer, nor any experimental report of this.Indeed the related application U.S. Pat. No. 7,169,272, which doesreport experimental formation of lipid bilayers in the same type ofdevice, discloses the supported lipid bilayers being chemically attacheddirectly on the electrodes. This uses similar techniques to thosepresented in Osman et al. cited above and suffers from the samedrawbacks relating to the lack of a sufficiently high resistive seal forstochastic measurements and the lack of an ionic reservoir for recordingionic flow across the bilayer system.

To summarize, the technologies described above either present methods ofbilayer formation which can not reproducibly achieve high resistance, orsuffer from low ionic reservoirs and are not capable of high durationdirect current measurements, or require a separate fluidic chamber foreach array element, limiting the scale up of that device to ahigh-density array. It would be desirable to reduce these problems.

BRIEF SUMMARY OF THE DISCLOSURE

According to a first aspect of the present disclosure, there is provideda method of forming a layer separating two volumes of aqueous solution,the method comprising:

(a) providing an apparatus comprising elements defining a chamber, theelements including a body of non-conductive material having formedtherein at least one recess opening into the chamber, the recesscontaining an electrode;

(b) applying a pre-treatment coating of a hydrophobic fluid to the bodyacross the recess;

(c) flowing aqueous solution, having amphiphilic molecules addedthereto, across the body to cover the recess so that aqueous solution isintroduced into the recess from the chamber and so that a layer of theamphiphilic molecules forms across the recess separating a volume ofaqueous solution introduced into the recess from the remaining volume ofaqueous solution.

Such a method allows the formation of layers of amphiphilic moleculeswhich are of sufficiently high quality for sensitive techniques such asstochastic sensing whilst using apparatus and techniques which arestraightforward to implement.

The apparatus used is relatively simple, and comprises a body ofionically non-conductive material having formed therein at least onerecess. It has been demonstrated, surprisingly, that it is possible toform a layer of the amphiphilic molecules across such a recess simply byflowing the aqueous solution across the body to cover the recess. Toachieve this, a pre-treatment coating of a hydrophobic fluid is appliedto the body across the recess. The pre-treatment coating assistsformation of the layer. The layer is formed without any need for acomplicated apparatus involving two chambers separated by a septum andrequiring a complicated fluidics arrangement to achieve separatefilling. This is because the method does not require the recess to bepre-filled prior to introducing aqueous solution into the chamber above.Instead, the aqueous solution is introduced into the recess from thechamber. Despite this, it is still possible to form the layer by merecontrol of the aqueous solution flowing into the chamber. Such flowcontrol is a straightforward practical technique.

In some embodiments, the method may allow the formation of layers ofamphiphilic molecules which are suitable for high sensitivity biosensorapplications such as stochastic sensing and single channel recording. Ithas been demonstrated possible to form layers of high resistanceproviding highly resistive electrical seals, having an electricalresistance of 1 GΩ or more, typically at least 100 GΩ, which, forexample, enable high-fidelity stochastic recordings from single proteinpores. In some embodiments, this maybe achieved whilst trapping a volumeof aqueous solution in the recess between the layer and the electrode.This maintains a significant supply of electrolyte. For example, thevolume of aqueous solution is sufficient to allow stable continuous docurrent measurement through membrane proteins inserted in the layer.This contrasts significantly with the known techniques described aboveusing supported lipid bilayers.

Furthermore, the simple construction of the apparatus allows theformation of a miniaturized apparatus having an array of plural recessesand allowing the layer across each recess to be electrically isolatedand individually addressed using its own electrode, such that theminiaturized array is equivalent to many individual sensors measuring inparallel from a test sample. The recesses may be relatively denselypacked, allowing a large number of layers to be used for a given volumeof test sample. Individual addressing may be achieved by providingseparate contacts to each electrode which is simple using modernmicrofabrication techniques, for example lithography.

Furthermore, in some embodiments, the method may allow the formation ofmultiple layers of one or more amphiphilic molecules within a singleapparatus across the plural recesses in an array using a verystraightforward technique.

In most applications, one or more membrane proteins may be subsequentlyinserted into the layer. Certain membrane proteins that may be used inaccordance with the disclosure are discussed in more detail below.

According to further aspects of the disclosure, there is provided anapparatus suitable for implementing such methods of formation of a layerof amphiphilic molecules.

Further details and features of the disclosure will now be described.

The amphiphilic molecules are typically a lipid. In some embodiments,the layer is a bilayer formed from two opposing monolayers of lipid. Thelipids may comprise one or more lipids. The lipid bilayer may alsocontain additives that affect the properties of the bilayer. Certainlipids and other amphiphilic molecules, and additives that can be usedin accordance with the disclosure are discussed in more detail below.

Various techniques may be applied to add the amphiphilic molecules tothe aqueous solution.

A first technique is simply to add the amphiphilic molecules to theaqueous solution outside the apparatus before introducing the aqueoussolution into the chamber.

A second technique comprises before introducing the aqueous solutioninto the chamber, to deposit the amphiphilic molecules on an internalsurface of the chamber, or elsewhere in the flow path of the aqueoussolution, for example in a fluidic inlet pipe connected to the inlet. Inthis case, the aqueous solution covers the internal surface during stop(c) whereby amphiphilic molecules are added to the aqueous solution. Inthis manner the aqueous solution is used to collect the amphiphilicmolecules from the internal surface. Such deposition of the amphiphilicmolecules has several advantages. It allows the formation of layer ofamphiphilic molecules in the absence of large amounts of organicsolvent, as would typically be present if the amphiphilic molecules wereadded directly to the aqueous solution. This means that it is notnecessary to wait for evaporation of the organic solvent before thelayer can be formed. In addition, this means that the apparatus is notrequired to be made from materials that are insensitive to organicsolvents. For instance, organic-based adhesives can be used andscreen-printed conductive silver/silver chloride paste can be used toconstruct electrodes.

In some embodiments, the deposited amphiphilic molecules may be dried.In such embodiments, an aqueous solution may be used to rehydrate theamphiphilic molecules. This allows amphiphilic molecules to be stablystored in the apparatus before use. In some embodiments, it also avoidsthe need for wet storage of amphiphilic molecules. Such dry storage ofamphiphilic molecules increases shelf life of the apparatus.

Several techniques may be used to insert a membrane protein into thelayer of amphiphilic molecules.

A first technique is simply for the aqueous solution to have a membraneprotein added thereto, whereby the membrane protein is insertedspontaneously into the layer of amphiphilic molecules. One or moremembrane protein(s) may be added to the aqueous solution outside theapparatus before introducing the aqueous solution into the chamber.Alternatively a membrane protein may be deposited on an internal surfaceof the chamber before introducing the aqueous solution into the chamber.In this case, the aqueous solution covers the internal surface duringstep (c), whereby one or more membrane protein(s) is added to theaqueous solution.

A second technique is for the aqueous solution to have vesiclescontaining a membrane protein added thereto, whereby the membraneprotein is inserted on fusion of the vesicles with the layer ofamphiphilic molecules.

A third technique is to insert one or more membrane protein by carryingthe membrane protein to the layer on a probe, for example an agar-tippedrod.

To form a layer of amphiphilic molecules, the aqueous solution is flowedacross the body to cover the recess. Formation is improved if amulti-pass technique is applied in which aqueous solution covers anduncovers the recess at least once before covering the recess for a finaltime. This is thought to be because at least some aqueous solution isleft in the recess which assists formation of the layer in a subsequentpass.

The pre-treatment coating is a hydrophobic fluid which assists formationof the layer by increasing the affinity of the amphiphilic molecules tothe surface of the body around the recess. In general any pre-treatmentthat modifies the surface of the surfaces surrounding the aperture toincrease its affinity to lipids may be used. Certain exemplary materialsfor the pre-treatment coating that may be used in accordance with thedisclosure are discussed in more detail below.

To assist in the spreading of the pre-treatment coating, surfacesincluding either or preferably both of (a) the outermost surface of thebody around the recess and (b) at least an outer part of the internalsurface of the recess extending from the rim of the recess may behydrophobic. This may be achieved by making the body with an outermostlayer formed of a hydrophobic material.

Another way to achieve this is for the surfaces to be treated by afluorine species, such as a fluorine radical, for example by treatmentwith a fluorine plasma during manufacture of an apparatus of thedisclosure.

The application of the pre-treatment coating may leave excesshydrophobic fluid covering said electrode contained in the recess. Thispotentially insulates the electrode by reducing ionic flow, therebyreducing the sensitivity of the apparatus in measuring electricalsignals. However various different techniques may be applied to minimizethis problem.

A first technique may comprise applying a voltage across an electrode ina recess and a further electrode in the chamber sufficient to reduce theamount of excess hydrophobic fluid covering said electrode contained inthe recess. This produces a similar effect to electro-wetting. Thevoltage is applied after flowing aqueous solution across the body tocover the recess so that aqueous solution flows into the recess. As thevoltage will rupture any layer formed across the recess, subsequentlythe aqueous solution is flowed to uncover the recess, and then aqueoussolution, having amphiphilic molecules added thereto, is flowed acrossthe body to re-cover the recess so that a layer of the amphiphilicmolecules forms across the recess.

A second technique may comprise making an inner part of the internalsurface of the recess hydrophilic. Typically this may be applied incombination with making the outer part of the internal surface of therecess hydrophobic. This may be achieved by making the body with aninner layer formed of a hydrophilic material and an outermost layerformed of a hydrophobic material.

A third technique may comprise providing on the electrode a hydrophilicsurface, for example a protective material, which repels the hydrophobicfluid applied in step (c) whilst allowing ionic conduction from theaqueous solution to the electrode. The protective material may be aconductive polymer, for example polypyrrole/polystyrene sulfonate.Alternatively, the protective material may be a covalently attachedhydrophilic species, such as thiol-PEG.

In general, a wide range of constructional features may be employed inthe apparatus to form the body of non-conductive material, the at leastone recess formed therein and the other elements defining the chamber.Examples are described in further detail below.

According to a second aspect of the present disclosure, there isprovided a method of improving the performance of an electrode in arecess in conducting electro-physiological measurements, the methodcomprising depositing a conductive polymer on the electrode.

Further according to a second aspect of the present disclosure, there isprovided an apparatus for conducting electro-physiological measurements,the apparatus comprising, a body having a recess in which an electrodeis located, wherein a conductive polymer is provided on the electrode.

It has been discovered that the providing a conductive polymer on anelectrode in a recess can improve the performance of the electrode inconducting electro-physiological measurements. One advantage is toimprove the electrode's performance as a stable electrode for conductingelectro-physiological measurements. A further advantage is to increasethe charge reservoir available to the electrode within the recesswithout increasing the volume of aqueous solution contained in therecess.

To allow better understanding, embodiments of the present disclosurewill now be described by way of non-limitative example with reference tothe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the disclosure may be understood by referring, inpart, to the present disclosure and the accompanying drawings, wherein:

FIG. 1 is a perspective view of an apparatus;

FIG. 2 is a cross-sectional view of the apparatus of FIG. 1, taken alongline II-II in FIG. 1, and showing the introduction of an aqueoussolution;

FIG. 3 is a cross-sectional view of the apparatus, similar to that ofFIG. 2 but showing the apparatus full of aqueous solution;

FIG. 4 is sequence of a cross-sectional, partial views of the recess inthe apparatus over an electrochemical electrode modification process;

FIG. 5 is an SEM image of a recess formed by CO₂ laser drilling;

FIG. 6 is an OM image of a recess formed using photolithography;

FIGS. 7A and 7B are 3D and 2D LP profiles, respectively, of a recessfunned using photolithography;

FIGS. 8A and 8B are 3D and 2D LP profiles, respectively, of a recessformed using photolithography, after electroplating;

FIG. 9 is a cross-sectional, partial view of the recess in the apparatuswith a pre-treatment coating applied;

FIGS. 10A, 10B, 10C, 10D and 10E are a sequence of cross-sectional,partial view of the recess in the apparatus during a method of removingexcess pre-treatment coating;

FIG. 11 is a cross-sectional, partial view of the recess in theapparatus having plural further layers in the body;

FIG. 12 is a diagram of an electrical circuit;

FIG. 13 is a perspective view of the apparatus and electrical circuitmounted on a printed circuit board;

FIG. 14 is a diagram of an electrical circuit for acquiring pluralsignals in parallel;

FIG. 15 is a graph of the applied potential and current response for adry apparatus;

FIG. 16 is a graph of the applied potential and current response for awet apparatus;

FIG. 17 is a graph of the applied potential and current response onelectro-wetting of the apparatus;

FIG. 18 is a graph of the applied potential and current response onformation of a layer of amphiphilic molecules;

FIGS. 19, 20, 21 and 22 are graphs of the applied potential and currentresponse for various different apparatuses;

FIGS. 23, 24 and 25 are plan views of a further layer in a modifiedapparatus having plural recesses;

FIGS. 26, 27 and 28 are plan views of the substrate in the modifiedapparatuses having plural recesses;

FIGS. 29 and 30 are graphs of the current response for two differentapparatuses having plural recesses;

FIG. 31 is a cross-sectional view of a portion of a modified apparatus;

FIG. 32 is a cross-sectional view of another modified apparatus;

FIG. 33 is a flow chart of a method of manufacture of the apparatus;

FIGS. 34A and 34B are 3D- and 2D surface profiles of a recess having anelectrode modified by electropolymerisation of polypyrrole, measured byprofilometry;

FIG. 35 is a graph of current recorded on an array of recesses having anelectrode modified by electropolymerisation of polypyrrole.

DETAILED DESCRIPTION OF THE DISCLOSURE

An apparatus 1 which may be used to form a layer of amphiphilicmolecules is shown in FIG. 1. The apparatus 1 includes a body 2 havinglayered construction as shown in FIGS. 2 and 3 comprising a substrate 3of non-conductive material supporting a further layer 4 also ofnonconductive material. In the general case, there may be plural furtherlayers 4, as described further below.

A recess 5 is formed in the further layer 4, in particular as anaperture which extends through the further layer 4 to the substrate 3.In the general case, there may be plural recesses 5, as describedfurther below.

The apparatus 1 further includes a cover 6 which extends over the body2. The cover 6 is hollow and defines a chamber 7 which is closed exceptfor an inlet 8 and an outlet 9 each formed by openings through the cover6. The lowermost wall of the chamber 7 is formed by the further layer 4in FIG. 2, but as an alternative the further layer 4 could be shaped toprovide side walls.

As described further below, in use aqueous solution 10 is introducedinto the chamber 7 and a layer 11 of amphiphilic molecules is formedacross the recess 5 separating aqueous solution 10 in the recess 5 fromthe remaining volume of aqueous solution in the chamber 7. The apparatusincludes the following electrode arrangement to allow measurement ofelectrical signals across the layer 11 of amphiphilic molecules.

Use of a chamber 7 which is closed makes it very easy to flow aqueoussolution 10 into and out of the chamber 7. This is done simply byflowing the aqueous solution 10 through the inlet 8 as shown in FIG. 2until the chamber 7 is full as shown in FIG. 3. During this process, gas(typically air) in the chamber 7 is displaced by the aqueous solution 10and vented through the outlet 9. For example, a simple fluidics systemattached to the inlet 8 may be used. This may be as simple as a plunger,although more complicated systems may be used to improve the control.However, the chamber 7 is not necessarily closed and may be open, forexample by forming the body 2 as a cup.

The substrate 3 has a first conductive layer 20 deposited on the uppersurface of the substrate 3 and extending under the further layer 4 tothe recess 5. The portion of the first conductive layer 20 underneaththe recess 5 constitutes an electrode 21 which also forms the lowermostsurface of the recess 5. The first conductive layer 20 extends outsidethe further layer 4 so that a portion of the first conductive layer 20is exposed and constitutes a contact 22.

The further layer 4 has a second conductive layer 23 deposited thereonand extending under the cover 6 into the chamber 7, the portion of thesecond conductive layer 23 inside the chamber 7 constituting anelectrode 24. The second conductive layer 23 extends outside the cover 6so that a portion of the second conductive layer 23 is exposed andconstitutes a contact 25.

The electrodes 21 and 24 make electrical contact with aqueous solutionin the recess 5 and chamber 7. This allows measurement of electricalsignals across the layer 11 of amphiphilic molecules by connection of anelectrical circuit 26 to the contacts 22 and 25. The electrical circuit26 may have basically the same construction as a conventional circuitfor performing stochastic sensing across a lipid bilayer formed in aconventional cell by the Montal & Mueller method.

An example design of the electrical circuit 26 is shown in FIG. 12. Theprimary function of the electrical circuit 26 is to measure theelectrical current signal developed between the electrodes 21 and 24 toprovide a meaningful output to the user. This may be simply an output ofthe measured signal, but in principle could also involve furtheranalysis of the signal. The electrical circuit 26 needs to besufficiently sensitive to detect and analyze currents which aretypically very low. By way of example, an open membrane protein mighttypically pass current of 100 pA to 200 pA with a 1M salt solution.

In this implementation, the electrode 24 in the chamber 7 is used as areference electrode and the electrode 21 in the recess 5 is used as aworking electrode. Thus the electrical circuit 26 provides the electrode24 with a bias voltage potential relative to the electrode 21 which isitself at virtual ground potential and supplies the current signal tothe electrical circuit 26.

The electrical circuit 26 has a bias circuit 40 connected to theelectrode 24 in the chamber 7 and arranged to apply a bias voltage whicheffectively appears across the two electrodes 21 and 24.

The electrical circuit 26 also has an amplifier circuit 41 connected tothe electrode 21 in the recess 5 for amplifying the electrical currentsignal appearing across the two electrodes 21 and 24. Typically, theamplifier circuit 41 consists of a two amplifier stages 42 and 43.

The input amplifier stage 42 connected to the electrode 21 converts thecurrent signal into a voltage signal.

The input amplifier stage 42 may comprise transimpedance amplifier, suchas an electrometer operational amplifier configured as an invertingamplifier with a high impedance feedback resistor, of for example 500MΩ,to provide the gain necessary to amplify the current signal whichtypically has a magnitude of the order of tens to hundreds of picoamps.

Alternatively, the input amplifier stage 42 may comprise a switchedintegrator amplifier. This is preferred for very small signals as thefeedback element is a capacitor and virtually noiseless. In addition, aswitched integrator amplifier has wider bandwidth capability. However,the integrator does have a dead time due to the necessity to reset theintegrator before output saturation occurs. This dead time may bereduced to around a microsecond so is not of much consequence if thesampling rate required is much higher. A transimpedance amplifier issimpler if the bandwidth required is smaller. Generally, the switchedintegrator amplifier output is sampled at the end of each samplingperiod followed by a reset pulse. Additional techniques can be used tosample the start of integration eliminating small errors in the system.

The second amplifier stage 43 amplifies and filters the voltage signaloutput by the first amplifier stage 42. The second amplifier stage 43provides sufficient gain to raise the signal to a sufficient level forprocessing in a data acquisition unit 44. For example with a 500MΩfeedback resistance in the first amplifier stage 42, the input voltageto the second amplifier stage 43, given a typical current signal of theorder of 100 pA, will be of the order of 50 mV, and in this case thesecond amplifier stage 43 must provide a gain of 50 to raise the 50 mVsignal range to 2.5V.

The electrical circuit 26 includes a data acquisition unit 44 which maybe a microprocessor running an appropriate program or may includededicated hardware. The data acquisition unit 44 may be a card to beplugged into a computer 45 such as a desktop or laptop. In this case,the bias circuit 40 is simply formed by an inverting amplifier suppliedwith a signal from a digital-to-analog converter 46 which may be eithera dedicated device or a part of the data acquisition unit 44 and whichprovides a voltage output dependent on the code loaded into the dataacquisition unit 44 from software. Similarly, the signals from theamplifier circuit 41 are supplied to the data acquisition card 40through an analog-to-digital converter 47.

The various components of the electrical circuit 26 may be formed byseparate components or any of the components may be integrated into acommon semiconductor chip. The components of the electrical circuit 26may be formed by components arranged on a printed circuit board. Anexample of this is shown in FIG. 13 wherein the apparatus 1 is bonded toa printed circuit board 50 with aluminum wires 51 connecting from thecontacts 22 and 25 to tracks 52 on the printed circuit board. A chip 53incorporating the electrical circuit 26 is also bonded to the printedcircuit board 50. Alternatively the apparatus 1 and the electricalcircuit 26 may be mounted on separate printed circuit boards.

In the case that the apparatus 1 contains plural recesses 5, each havinga respective electrode 21, then the electrical circuit 26 is modifiedessentially by replicating the amplifier circuit 41 and A/D converter 47for each electrode 21 to allow acquisition of signals from each recess 5in parallel. In the case that the input amplifier stage 42 comprisesswitched integrators then those would require a digital control systemto handle the sample-and-hold signal and reset integrator signals. Thedigital control system is most conveniently configured on afield-programmable-gate-array device (FPGA). In addition the FPGA canincorporate processor-like functions and logic required to interfacewith standard communication protocols i.e. USB and Ethernet.

FIG. 14 shows a possible architecture of the electrical circuit 26 andis arranged as follows. The respective electrodes 21 of the apparatus 1are connected to the electrical circuit 26 by an interconnection 55, forexample the aluminum wires 51 and the printed circuit board in thearrangement of FIG. 13. In the electrical circuit 26, the amplifiercircuits 41 may be formed in one or more amplifier chips 56 havingplural channels. The signals from different electrodes 21 may be onseparate channels or multiplexed together on the same channel. Theoutputs of the one or more amplifier chips 56 are supplied via the A/Dconverter 47 to a programmable logic device 57 for receiving the signalon each channel. For example to handle signals from an apparatus having1024 recesses, the programmable logic device 57 might operate at a speedof the order of 10 Mbits/s. The programmable logic device 57 isconnected via an interface 58, for example a USB interface, to acomputer 59 to supply the signals to the computer 59 for storage,display and further analysis.

During use the apparatus 1 may be enclosed in a Faraday cage to reduceinterference.

Various materials for the components of the apparatus 1 will now bediscussed. The materials for each component of apparatus 1 aredetermined by the properties required to enable the component tofunction correctly during operation, but the cost and manufacturingthroughput are also considered. All materials may be chosen to providesufficient mechanical strength to allow robust handling, and surfacescompatible with bonding to the subsequent layers.

The material of the substrate 3 is chosen to provide a rigid support forthe remainder of the apparatus 1. The material is also chosen to providea high resistance and low capacitance electrical insulation betweenadjacent electrodes 21 when there are plural recesses 5. Possiblematerials include without limitation: polyester (e.g. Mylar), or anotherpolymer; or silicon, silicon nitride, or silicon oxide. For example, thesubstrate may comprise a silicon wafer with a thermally grown oxidesurface layer.

The material of the further layer 4 (or in the general case layers) arechosen to provide a high resistance and low capacitance electricalinsulation between the electrodes 21 and 24 and also, when there areplural recesses 5, between the electrodes 21 and 24 of adjacent recesses5. Also the surface of the further layer 4 should be chemically stableboth to the pre-treatment coating applied before operation (as discussedbelow) and to the aqueous solution 10. Lastly, the further layer 4should be mechanically robust in order to maintain its structuralintegrity and coverage of the first conductive layer 20, and should besuitable for subsequent attachment of the cover 6.

The following is a list of possible materials for the further layer 4,together with thicknesses which have been successfully employedexperimentally, although these thicknesses are not limitative:photoresist (e.g. SUB photoresist or Cyclotene) with a variety ofthicknesses; polycarbonate, 6 μm thick film; PVC, 7 μm thick film;polyester, 50 μm thick film; adhesive backed polyester, 25 μm and 50 μmthick film; thermal laminating films, e.g. Magicard 15 μm thick andMurodigital 35 μm; or a screen-printed dielectric ink.

Advantageously, surfaces including (a) the outermost surface of the body2 around the recess and (b) the outer part of the internal surface ofthe recess 5 extending from the rim of the recess 5 are hydrophobic.This assists in the spreading of the pre-treatment coating and thereforealso formation of a lipid bilayer. One particular way to achieve this isto modify these surfaces by a fluorine species. Such a fluorine speciesis any substance capable of modifying the surfaces to provide afluorine-containing layer. The fluorine species is preferably onecontaining fluorine radicals. For example the modification may beachieved by treating the body 2 with a fluorine plasma, for example aCF₄ during manufacture.

The conductive layers 20 and 23 will now be discussed further.

The material of the electrodes 21 and 24 should provide anelectrochemical electrode in contact with the aqueous solution 10,enabling measurement of low currents, and should be stable to thepre-treatment coating and aqueous solution 10. The material of theremainder of the conductive layers 20 and 23 (usually but notnecessarily the same as the electrodes 21 and 24) also provideselectrical conductance from the electrodes to the contacts 22 and 25.The first conductive layers 20 will also accept bonding of the furtherlayers 4. The conductive layers 20 and 23 can be constructed with pluraloverlapping layers and/or an appropriate surface treatment. One possiblematerial is platinum, coated with silver at the area exposed to the testsolution and then silver chloride formed on top of the silver. Possiblematerials for the first conductive layer 20 include without limitation:Silver/silver chloride electrode ink; silver with or without a surfacelayer, for example of silver chloride formed by chloridisation or ofsilver fluoride formed by fluoridisation; gold with or without redoxcouple in solution; platinum with or without redox couple in solution;ITO with and without redox couple in solution; gold electrochemicallycoated with conductive polymer electrolyte; or platinumelectrochemically coated with conductive polymer electrolyte. Possiblematerials for the second conductive layer 23 include without limitation:silver/silver chloride electrode ink; silver wire; or chloridised silverwire.

Some specific examples of include: the substrate 3 being silicon and theconductive layer 20 being a metal conductor (diffusion or polysiliconwires are poor methods) buried in a silicon oxide insulating layer (e.g.using typical semiconductor fabrication technology); the substrate 3being glass and the conductive layer 20 being metal conductors (e.gusing typical LCD display technology); or the substrate 3 being apolymeric substrates and the conductive layer 20 being an ablated metalor printed conductor (e.g. using typical glucose biosensor technology).

The requirements for the material of the cover 6 are to be easilyattached to create a seal for the chamber 7, to be compatible with boththe pre-treatment coating and the aqueous solution 10. The following arepossible materials, together with thicknesses which have beensuccessfully employed experimentally, although these thicknesses are notlimitative: silicone rubber, 0.5, 1.0, 2.0 mm thick; polyester, 0.5 mmthick; or PMMA (acrylic) 0.5 mm to 2 mm thick.

Various methods of manufacturing the apparatus 1 will now be discussed.In general terms, the layered construction of the apparatus 1 is simpleand easy to form by a variety of methods. Three different fabricationtechnologies which have actually been applied are: lamination of polymerfilms; printed circuit board manufacture with high resolution soldermask formation and photolithography using silicon wafers or glass.

An example of a lamination process is as follows.

The substrate 3 is a 250 μm thick polyester sheet (Mylar), and the firstconductive layer 20 is deposited by either: screen printingsilver/silver chloride electrode ink; adhesion of metal foil; or vapourdeposition (sputtering or evaporation). The further layer 4 is thenlaminated onto the substrate 3 by either: a pressure-sensitive adhesive;a thermally activated adhesive; or using the wet silver/silver chlorideink as the adhesive painted directly onto the dielectric beforelamination (referred to as “painted electrodes”). The aperture in thefurther layer 4 that forms the recess 5 is created with 5-100 μmdiameter either before or after lamination to the substrate 3 by either:electrical discharge (sparking); or laser drilling, for example by anexcimer, solid state or CO₂ laser. An apparatus created by lamination ofpolymer films sometimes requires an additional sparking step to activatethe electrodes prior to use. The second conductive layer 23 is formed ontop of the further layer 4 by screen printing. The cover 6 is laminatedon top using pressure sensitive adhesive.

An example of a process employing photolithography using silicon wafersis as follows.

The substrate 3 is a silicon wafer with an oxide surface layer. Thefirst conductive layer 20 is formed by gold, silver, chloridised silver,platinum or ITO deposited onto the substrate 3. Photoresist (e.g. SUB)is then spin-coated over the substrate 3 to form the further layer 4.The recess 5 is formed with 5-100 μm diameter by removal of thephotoresist following UV exposure using a mask to define the shape ofthe recess 5. The second conductive layer 23 is formed on top of thefurther layer 4, for example by screen printing. The cover 6 islaminated on top using pressure sensitive adhesive.

The ability to use this type of process is significant because it allowsthe apparatus to be formed on silicon chips using standard silicon waferprocessing technology and materials.

The electrodes 21 and 24 will now be discussed further.

For stable and reliable operation, the electrodes 21 and 24 shouldoperate at the required low current levels with a low over-potential andmaintain their electrode potential value over the course of themeasurement. Further, the electrodes 21 and 24 should introduce aminimum amount of noise into the current signal. Possible materials forthe electrodes 21 and 24 include without limitation: Silver/silverchloride electrode ink; silver with or without a surface layer, forexample of silver chloride formed by chloridisation or of silverfluoride formed by fluoridisation; gold with or without redox couple insolution; platinum with or without redox couple in-solution; ITO withand without redox couple in solution; palladium hydride, goldelectrochemically coated with conductive polymer electrolyte; orplatinum electrochemically coated with conductive polymer electrolyte.

Silver is a good choice for the material of electrodes 21 and 24 but isdifficult to incorporate in a silicon wafer manufacturing process due toits tendency to undergo oxidation on exposure to light, air and hightemperatures. To avoid this problem it is possible to manufacture theapparatus with an inert conductive material (e.g. Pt or Au) in therecess, and then change the surface type or properties of the inertconductive material using methods including but not limited toelectroplating, electropolymerisation, electroless plating, plasmamodification, chemical reaction, and other coating methods known in theart.

Electroplating of silver may be achieved, for example, using a modifiedversion of the method of Polk et al., “Ag/AgCl microelectrodes withimproved stability for microfluidics”, Sensors and Actuators B 114(2006) 239-247. A plating solution is prepared by addition of 0.41 g ofsilver nitrate to 20 ml of 1M ammonium hydroxide solution. This israpidly shaken to avoid precipitation of the insoluble silver oxide, andto facilitate the formation of the diammine silver complex. The solutionis always fresh to avoid fall in plating efficiency. The plating isperformed using conventional equipment, connecting the electrode 21 asthe cathode and using a platinum electrode is used as the anode. Forexample in the case of plating on Pt electrodes, a potential of −0.58Vis applied to the cathode, with the anode being held at groundpotential, whereas in the case of plating on Au electrodes, thepotential is held at −0.48V with respect to ground. A target charge of5.1×10³C/m² has been found empirically to result in a silver depositionof between 1 μm and 2 μm for a 100 μm diameter electrode, typicallytaking of the order of 60 s.

In performing such plating it is desirable to achieve uniformpenetration of the aqueous plating solution to the bottom of the recess5. In the case that the layer 4 is formed from a naturally hydrophobicmaterial (e.g. SUS photoresist) and in order to ensure uniform wettingof the recess, desirably the degree of hydrophilicity can be increased.Three methods to achieve this are as follows. A first method isapplication of a lipid to the surface of the layer 4, so that the lipidacts as a surfactant, facilitating the entry of the plating solution. Asecond method is exposure of the layer 4 to oxygen plasma whichactivates the material of the layer and produces hydrophilic functionalgroups. This produces a well defined hydrophilic and clean surface. Athird method is to add ethanol to the plating solution.

Where the electrode 21 is made of silver (or indeed other metals), theouter surface of the electrode is desirably converted to a halide, inorder for the electrode 21 to function efficiently as a provider of astable reference potential. In common usage, the halide used ischloride, since the conversion of silver to silver chloride isrelatively straightforward to achieve, for example by electrolysis in asolution of hydrochloric acid. Alternative chemical methods avoiding theuse of a potentially corrosive acid which may affect the surfacecondition of the layer 4 include a) sweeping voltammetry in 3M sodiumchloride solution, and b) a chemical etching by immersion of theelectrode 21 in 50 mM ferric chloride solution.

An alternative halogen for the halidisation is fluorine. The choice offluorine has the significant advantage that the silver fluoride layercan be formed in the same step as modification of surfaces (a) and (b)of the body 2 to make them hydrophobic, as discussed above. For examplethis may be achieved during manufacture of the apparatus 1 by treatmentof the body 2 by a fluorine plasma for example a CF₄ plasma. This iseffective to modify the surfaces of the body 2, particularly in the casethat the layer 4 is a photoresist such as SU8 to achieve a sufficientdegree of hydrophobicity to support the formation of a stable lipidbilayer. At the same time exposure to the fluorine plasma converts themetal of the electrode 21 into an outer layer of metal fluoride.

There will now be discussed some possible adaptations of the electrode21 in the recess 5 as alternatives to the use of a fluorine plasma asdiscussed above.

The electrode 21 may be electrochemically modified to change thesurface-type. This allows use of additional materials with good bulkproperties but poor surface properties, such as gold. Possibleelectrochemical surface modifications include without limitation: silverelectroplating; electrochemical chloridisation of silver;electropolymerisation of a polymer/polyelectrolyte.

By way of further example, one possible sequence of modification isshown in FIG. 4 in which a coating 37 of silver is formed on theelectrode 21 formed of gold or platinum by electrochemical deposition.Electroplating may typically be performed in 0.2M AgNO₂, 2M KI, 0.5 mMNa₂S₂O₃ at −0.48V using a standard single liquid junction Ag/AgClreference electrode and a platinum counter electrode. A typicalthickness of the coating 37 is estimated to be 750 nm with a depositiontime of about 50 s and about 50 μC charge passed. Subsequently achloridised layer 38 is formed by chloridisation, typically at +150 mVin 0.1M HCl for 30 s.

Another possible surface modification is to apply a conductive polymer.The conductive polymer may be any polymer which is conductive. Asuitable conductive polymer will have mobile charge carriers. Typicallysuch a conductive polymer will have a backbone having delocalisedelectrons which are capable of acting as charge carriers, allowing thepolymer to conduct. The conductive polymer may be doped to increase itsconductivity, for example by a redox process or by electrochemicaldoping. Suitable conductive polymers include, without limitation:polypyrroles, polyacetylenes, polythiophenes, polyphenylenes,polyanilines, polyfluorenes, poly(3-alkylthiophene)s,polytetrathiafulvalenes, polynaphthalenes, poly(p-phenylene sulfide)s,polyindoles, polythionines, polyethylenedioxythiophenes, andpoly(para-phenylene vinylene)s.

One possible conductive polymer is a polypyrrole, which may be doped,for example with polystyrene sulfonate. This may be deposited, forexample, on an electrode 21 of gold by electrooxidizing an aqueoussolution of 0.1M pyrrole+90 mM polystyrene sulfonate in 0.1M KCl at+0.80V vs. Ag/AgCl reference electrode. The estimated thickness ofpolymer deposited is 1 μm at 30 μC, based on an assumption that 40mC/cm² of charge produces a film of thickness around 0.1 μm. Thepolymerization process can be represented as follows, where PE standsfor polystyrene sulfonate:

One advantage of using a conductive polymer deposited on an inertelectrode, such as polypyrrole doped with polystyrene,electropolymerised onto gold or platinum, is to improve the electrode'sperformance as a stable electrode for conducting electrophysiologicalmeasurements. A further advantage is to increase the charge reservoiravailable to the electrode within the recess without increasing thevolume of aqueous solution contained in the recess. These advantages aregenerally applicable when conducting electrophysiological measurementsusing an electrode in a recess, such as the electrode 21 in theapparatus 1.

Other advantages of using a conductive polymer on the electrode 21 inthe recess 5 of the apparatus 1 include but are not limited to controlof the hydrophilic nature of the electrode surface to aid wetting of theelectrode surface by the aqueous buffer solution and similarlyprevention of blocking of the electrode by the chemical pre-treatmentprior to bilayer formation.

FIGS. 34A and 34B are 3D and 2D surface profiles of an example electrodemodified by electropolymerisation of polypyrrole, measured byprofilometry. The thickness of electrochemically deposited polymer filmin this example is about 2 μm. FIG. 35 shows the current recorded on anarray of recesses modified by electropolymerisation of polypyrrole,showing stable lipid bilayers and single molecule detection ofcyclodextrin from inserted protein pores.

In all embodiments, an alternative to the second conductive layer 23 isto form an electrode in the chamber 7 simply by insertion through thecover 6 of a conductive member, such as a chloridised silver wire.

In order to characterise the electrodes 21, visualisation of recesses 5formed in a body 2 has been conducted using optical microscopy (OM),scanning electron microscopy (SEM), and laser profilometry (LP).

FIG. 5 shows an SEM image of a recess 5 formed by drilling with a CO₂laser in an apparatus 1 formed by lamination of polymer layers, withsubsequent application of electrical discharge to activate the electrode21. The image illustrates that the geometry of the recess 5 is poorlydefined using this method of formation, with considerable surface damagetherearound and variability in diameter, although it is hoped this maybeimproved through optimisation of the laser characteristics.

FIG. 6 shows an OM image of a recess 5 formed using photolithography ofa further layer 4 of SU8 photoresist over an electrode 21 of vapourdeposited gold on a substrate 3 of silicon. Similarly, FIGS. 7A and 7Bare 3D and 2D LP profiles of a similarly manufactured recess 5. FIGS. 8Aand 8B are 3D and 2D LP profiles of the same recess 5 afterelectroplating to form a coating 38 of silver. These images show thatphotolithography provides a high degree of control of the geometry anddimensions of the recess.

Excimer laser methods also produce a controlled geometry similar tophotolithography.

There will now be described an example of a method of manufacture of theapparatus 1, as shown in FIG. 33. The rationale of this method is toprovide high throughput manufacture. This is achieved by processing awafer of silicon which forms the substrate 3 of plural apparatuses 1 andwhich is subsequently diced. The wafer is prepared with an insulatinglayer, for example a thermally grown silicon-oxide.

First the wafer is prepared. In step S1, the wafer is cleaned. In stepS2, the wafer is subjected to a BF dif to improve adhesion of metals andresist. Typical conditions are a 3 minute dip in 10:1 buffered oxideetch. In S3, the wafer is subjected to a bake as a dehydration step.Typical conditions are baking for 1 hour at 200° C. in an oven.

Next, the wafer is metallised to provide the first conductive layer 20of each apparatus 1. In step S4, photoresist is spun onto the waferwhich is then subjected to UV light to form the desired pattern. In stepS5, the conductive layers 20 are deposited, for example consisting ofsuccessive layers of Cr and Au. Typically of respective thicknesses 50nm and 300 nm. In step S6 the resist is removed for example by soakingin acetone.

Next, the layers 4 and recesses 5 are formed. In step S7, photoresistadhesion is improved by the application of an O₂ plasma and dehydrationbake for example in an oven. In step S8, the wafer has applied theretophotoresist which is then subjected to UV exposure to form the layers 4and recesses, for example SU8-10 with a thickness of 20 m. In step S9 aninspection and measurement of the recesses is performed.

Next, the electrodes 21 are plated. In step S10, the surface is preparedfor plating by performing an O₂ plasma descum. In step S11, silverplating of the electrode is performed, as described above, for exampleto form a plating thickness of 1.5 μm.

In step S12, the wafer is diced to form the bodies 2 of separateapparatuses 1.

Lastly, the bodies 2 are treated by a CF₄ plasma which modifies thesurfaces of the body 2 and the electrode 21 as discussed above. Atypical exposure is for 12 minutes at 70 W and 160 mTorr.

In practice with an apparatus 1 manufactured using this method, theresults of bilayer formation and pore current stability have beencomparable to those achieved with bodies plated and chloridised by wetchemical means.

The method of using the apparatus 1 to form a layer 11 of amphiphilicmolecules will now be described. First the nature of the amphiphilicmolecules that may be used will be considered.

The amphiphilic molecules are typically a lipid. In this case, the layeris a bilayer formed from two opposing monolayers of lipid. The twomonolayers of lipids are arranged such that their hydrophobic tailgroups face towards each other to form a hydrophobic interior. Thehydrophilic head groups of the lipids face outwards towards the aqueousenvironment on each side of the bilayer. The bilayer may be present in anumber of lipid phases including, but not limited to, the liquiddisordered phase (fluid lamellar), liquid ordered phase, solid orderedphase (lamellar gel phase, interdigitated gel phase) and planar bilayercrystals (lamellar sub-gel phase, lamellar crystalline phase).

Any lipids that may form a lipid bilayer may be used. The lipids arechosen such that a lipid bilayer having the required properties, such assurface charge, ability to support membrane proteins, packing density ormechanical properties, is formed. The lipids can comprise one or moredifferent lipids. For instance, the lipids can contain up to 100 lipids.The lipids preferably contain 1 to 10 lipids. The lipids may comprisenaturally-occurring lipids and/or artificial lipids.

The lipids typically comprise a head group, an interfacial moiety andtwo hydrophobic tail groups which may be the same or different. Suitablehead groups include, but are not limited to, neutral head groups, suchas diacylglycerides (DG) and ceramides (CM); zwitterionic head groups,such as phosphatidyl choline (PC), phosphatidylethanolamine (PE) andsphingomyelin (SM); negatively charged head groups, such asphosphatidylglycerol (PG); phosphatidylserine (PS), phosphatidylinositol(PI), phosphatic acid (PA) and cardiolipin (CA); and positively chargedheadgroups, such as trimethylammonium-Propane (TAP). Suitableinterfacial moieties include, but are not limited to,naturally-occurring interfacial moieties, such as glycerol-based orceramide-based moieties. Suitable hydrophobic tail groups include, butare not limited to, saturated hydrocarbon chains, such as lauric acid(n-Dodecanolic acid), myristic acid (n-Tetradecononic acid), palmiticacid (n-Hexadecanoic acid), stearic acid (n-Octadecanoic) and arachidic(n-Eieosanoic); unsaturated hydrocarbon chains, such as oleic acid(cis-9-Octadecanoic); and branched hydrocarbon chains, such asphytanoyl. The length of the chain and the position and number of thedouble bonds in the unsaturated hydrocarbon chains can vary. The lengthof the chains and the position and number of the branches, such asmethyl groups, in the branched hydrocarbon chains can vary. Thehydrophobic tail groups can be linked to the interfacial moiety as anether or an ester.

The lipids can also be chemically-modified. The head group or the tailgroup of the lipids may be chemically-modified. Suitable lipids whosehead groups have been chemically-modified include, but are not limitedto, PEG-modified lipids, such as 1,2-Diacyl-sn-Glycero3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000];functionionalised PEG Lipids, such as 1,2-Distearoyl-sn-Glycero-3Phosphoethanolamine-N-[Biotinyl(Polyethylene Glycol)2000]; and lipidsmodified for conjugation, such as1,2-Dioleoyl-sn-Olycero-3-Phosphoethanolamine-N-(succinyl) and1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N.(Biotinyl). Suitablelipids whose tail groups have been chemically-modified include, but arenot limited to, polymerisable lipids, such as1,2-bis(10,12-tricosadiynoyl)-sn-Glycero-3-Phosphocholine; fluorinatedlipids, such as1-Palmitoyl-2-(16-Pluoropalmitoyl)-sn-Olycero-3-Phosphocholine;deuterated lipids, such as1,2-Dipalmitoyl-D62-sn-Glycero-3-Phosphocholine; and ether linkedlipids, such as 1,2-Di-O-phytanyl-sn-Glycero-3-Phosphocholine.

The lipids typically comprise one or more additives that will affect theproperties of the lipid bilayer. Suitable additives include, but are notlimited to, fatty acids, such as palmitic acid, myristic acid and oleicacid; fatty alcohols, such as palmitic alcohol, myristic alcohol andoleic alcohol; sterols, such as cholesterol, ergosterol, lanosterol,sitosterol and stigmasterol; lysophospholipids, such as1-Acyl-2-Hydroxy-sn-Glycero-3-Phosphocholine; and ceramides. The lipidpreferably comprises cholesterol and/or ergosterol when membraneproteins are to be inserted into the lipid bilayer.

However, although lipids are commonly used to form bilayers, it isexpected that in general the method is applicable to any amphiphilicmolecules which may form a layer.

As to the aqueous solution 10, in general a wide range of aqueoussolutions 10 that are compatible with the formation of a layer 11 ofamphiphilic molecules may be used. The aqueous solution 10 is typicallya physiologically acceptable solution. The physiologically acceptablesolution is typically buffered to a pH of 3 to 11. The pH of the aqueoussolution 10 will be dependent on the amphiphilic molecules used and thefinal application of the layer 11. Suitable buffers include withoutlimitation: phosphate buffered saline (PBS);N-2-Hydroxyethylpiperazine-N′-2-Ethanesulfonic Acid (HEPES) bufferedsaline; Piperazine-1,4-Bis-2-Ethanesulfonic Acid (PIPES) bufferedsaline; 3-(n-Morpholino)Propanesulfonic Acid (MOPS) buffered saline; andTris(Hydroxymethyl)aminomethane (TRIS) buffered saline. By way ofexample, in one implementation, the aqueous solution 10 may be 10 mM PBScontaining 1.0M sodium chloride (NaCl) and having a pH of 6.9.

The method of using the apparatus 1 is as follows.

First, a pre-treatment coating 30 is applied to the body 2 across therecess 5, as shown in FIG. 9. The pre-treatment coating 30 is ahydrophobic fluid which modifies the surface of the body 2 surroundingthe recess 5 to increase its affinity to the amphiphilic molecules.

The pre-treatment coating 30 is typically an organic substance, usuallyhaving long chain molecules, in an organic solvent. Suitable organicsubstances include without limitation: n-decane, hexadecane,isoecoisane, squalene, fluoroinated oils (suitable for use withfluorinated lipids), alkyl-silane (suitable for use with a glassmembrane) and alkyl-thiols (suitable for use with a metallic membrane).Suitable solvents include but are not limited to: pentane, hexane,heptane, octane, decane, and toluene. The material might typically be0.1 μl to 10 μl of 0.1% to 50% (v/v) hexadecane in pentane or anothersolvent, for example 2 μl of 1% (v/v) hexadecane in pentane or anothersolvent, in which case lipid, such asdiphantytanoyl-sn-glycero-3-phosphocholine (DPhPC), might be included ata concentration of 0.6 mg/ml.

Some specific materials for the pre-treatment coating 30 are set out inTable 1 by way of example and without limitation.

TABLE 1 Pre-treatment formulation Volumes applied 0.3% hexadecane inpentane 2x 1 μl 1% hexadecane in pentane 2x2x 0.5 μl; 2x 0.5 μl; 1 μl;2x 1 μl; 2x 1 μl; 2 μl; 2x 2 μl; 5 μl 3% hexadecane in pentane 2x 1 μl;2 μl 10% hexadecane in pentane 2x 1 μl; 2 μl; 5 μl 0.5% hexadecane + 0.6mg/ml 5 μl DPhPC lipid in pentane 1.0% hexadecane + 0.6 mg/ml 2x 2x 0.5μl DPhPC lipid in pentane 1.5% hexadecane + 0.6 mg/ml 2 μl; 2x 1 μlDPhPC lipid in pentane

The pre-treatment coating 30 may be applied in any suitable manner, forexample simply by capillary pipette. The pre-treatment coating 30 may beapplied before or after the cover 6 is attached to the apparatus 1.

The precise volume of material of the pre-treatment coating 30 requireddepends on the size of the recess 5, the formulation of the material,and the amount and distribution of the when it dries around theaperture. In general increasing the amount (by volume and/or byconcentration) improves the effectiveness, although excessive materialcan cover the electrode 21 as discussed below. As the diameter of therecess 5 is decreased, the amount of material of the pre-treatmentcoating 30 required also varies. The distribution of the pre-treatmentcoating 30 can also affect effectiveness, this being dependent on themethod of deposition, and the compatibility of the membrane surfacechemistry. Although the relationship between the pre-treatment coating30 and the ease and stability of layer formation is complex, it isstraightforward to optimize the amount by routine trial and error. Inanother method the chamber 7 can be completely filled by pre-treatmentin solvent followed by removal of the excess solvent and drying with agas flow.

The pre-treatment coating 30 is applied across the recess 5. As a resultand as shown in FIG. 9, the pre-treatment coating 30 covers the surfaceof the body 2 around the recess 5. The pre-treatment coating 30 alsoextends over the rim of the recess 5 and desirably covers at least theoutermost portion of the side walls of the recess 5. This assists withformation of the layer 11 of amphiphilic molecules across the recess 5.

However, the pre-treatment coating 30 also has a natural tendency duringapplication to cover the electrode 21. This is undesirable as thepre-treatment coating 30 reduces the flow of current to the electrode 21and therefore reduces the sensitivity of measurement of electricalsignals, in the worst case preventing any measurement at all. A numberof different techniques may be employed to reduce or avoid this problem,and will be discussed after the description of forming the layer 11 ofamphiphilic molecules.

After application of the pre-treatment coating 30, the aqueous solution10 is flowed across the body 2 to cover the recess 5 as shown in FIG. 3.This step is performed with the amphiphilic molecules added to theaqueous solution 10. It has been demonstrated that, with an appropriatepre-treatment coating 30 this allows the formation of the layer 11 ofamphiphilic molecules across the recess 5. Formation is improved if amulti-pass technique is applied in which aqueous solution 10 covers anduncovers the recess 5 at least once before covering the recess 5 for afinal time. This is thought to be because at least some aqueous solutionis left in the recess 5 which assists formation of the layer 11 in asubsequent pass. Notwithstanding this, it should be noted that theformation of the layer 11 is reliable and repeatable. This is despitethe fact that the practical technique of flowing aqueous solution 10across the body 2 through the chamber 7 is very easy to perform.Formation of the layer 11 may be observed by monitoring of the resultantelectrical signals across the electrodes 21 and 24, as described below.Even if a layer 11 fails to form it is a simple matter to performanother pass of the aqueous solution 10. Such reliable formation of alayer 11 of amphiphilic molecules using a simple method and a relativelysimple apparatus 1 is a particular advantage of the present disclosure.

Furthermore, it has been demonstrated that the layers 11 of amphiphilicmolecules are of high quality, in particular being suitable for highsensitivity biosensor applications such as stochastic sensing and singlechannel recording. The layers 11 have high resistance providing highlyresistive electrical seals, having an electrical resistance of 1 GΩ ormore, typically at least 100 GΩ which, for example, enableshigh-fidelity stochastic recordings from single protein pores.

This is achieved whilst trapping a volume of aqueous solution 10 in therecess 5 between the layer 11 and the electrode 21. This maintains asignificant supply of electrolyte. For example, the volume of aqueoussolution 10 is sufficient to allow stable continuous do currentmeasurement through membrane proteins inserted in the layer.Experimental results demonstrating these advantages are set out later.

There are various techniques for adding the amphiphilic molecules to theaqueous solution 10, as follows.

A first technique is simply to add the amphiphilic molecules to theaqueous solution 10 outside the apparatus 1 before introducing theaqueous solution 10 into the chamber 7.

A second technique which has particular advantage is, before introducingthe aqueous solution 10 into the chamber 7, to deposit the amphiphilicmolecules on an internal surface of the chamber 7, or on an internalsurface elsewhere in the flow path of the aqueous solution 10 into thechamber 7, for example in a fluidic inlet pipe connected to the inlet.The amphiphilic molecules can be deposited on any one or more of theinternal surfaces of the chamber 7, including a surface of the furtherlayer 4 or of the cover 6. The aqueous solution 10 covers the internalsurface during its introduction, whereby the amphiphilic molecules areadded to the aqueous solution 10. In this manner, the aqueous solution10 is used to collect the amphiphilic molecules from the internalsurface. The aqueous solution 10 may cover the amphiphilic molecules andthe recess 5 in any order but preferably covers the amphiphilicmolecules first. If the amphiphilic molecules are to be covered first,the amphiphilic molecules are deposited along the flow path between theinlet 8 and the recess 5.

Any method may be used to deposit the lipids on an internal surface ofthe chamber 7. Suitable methods include, but are not limited to,evaporation or sublimation of a carrier solvent, spontaneous depositionof liposomes or vesicles from a solution and direct transfer of the drylipid from another surface. An apparatus 1 having lipids deposited on aninternal surface may be fabricated using methods including, but notlimited to, drop coating, various printing techniques, spin-coating,painting, dip coating and aerosol application.

The deposited amphiphilic molecules are preferably dried. In this case,the aqueous solution 10 is used to rehydrate the amphiphilic molecules.This allows the amphiphilic molecules to be stably stored in theapparatus 1 before use. It also avoids the need for wet storage ofamphiphilic molecules. Such dry storage of amphiphilic moleculesincreases shelf life of the apparatus. Even when dried to a solid state,the amphiphilic molecules will typically contain trace amounts ofresidual solvent. Dried lipids are preferably lipids that comprise lessthan 50 wt % solvent, such as less than 40 wt %, less than 30 wt %, lessthan 20 wt %, less than 15 wt %, less than 10 wt % or less than 5 wt %solvent.

In most practical uses, a membrane protein is inserted into the layer 11of amphiphilic molecules. There are several techniques for achievingthis.

A first technique is simply for the aqueous solution 10 to have amembrane protein added thereto, whereby the membrane protein is insertedspontaneously into the layer 11 of amphiphilic molecules after a periodof time. The membrane protein may be added to the aqueous solution 10outside the apparatus 1 before introducing the aqueous solution 10 intothe chamber 7. Alternatively the membrane protein may be added afterformation of the layer 11.

Another way of adding the membrane protein to the aqueous solution 10 isto deposit it on an internal surface of the chamber 7 before introducingthe aqueous solution 10 into the chamber 7. In this case, the aqueoussolution 10 covers the internal surface during its introduction, wherebythe membrane protein is added to the aqueous solution 10 andsubsequently will spontaneously insert into layer 11. The membraneproteins may be deposited on any one or more of the internal surfaces ofthe chamber 7, including a surface of the further layer 4 or of thecover 6. The membrane proteins can be deposited on the same or differentinternal surface as the amphiphilic molecules (if also deposited). Theamphiphilic molecules and the membrane proteins may be mixed together.

Any method may be used to deposit the membrane proteins on an internalsurface of the chamber 7. Suitable methods include, but are not limitedto, drop coating, various printing techniques, spin-coating, painting,dip coating and aerosol application.

The membrane proteins are preferably dried. In this case, the aqueoussolution 10 is used to rehydrate the membrane proteins. Even when driedto a solid state, the membrane proteins will typically contain traceamounts of residual solvent. Dried membrane proteins are preferablymembrane proteins that comprise less than 20 wt % solvent, such as lessthan 15 wt %, less than 100 wt % or less than 5 wt % solvent.

A second technique is for the aqueous solution 10 to have vesiclescontaining the membrane protein added thereto, whereby the membraneprotein is inserted on fusion of the vesicles with the layer 11 ofamphiphilic molecules.

A third technique is to insert the membrane protein by carrying themembrane protein to the layer 11 on a probe, for example an agar-tippedrod, using the techniques disclosed in WO-2006/100484. Use of a probemay assist in selectively inserting different membrane proteins indifferent layers 11, in the case that the apparatus has an array ofrecesses. However, this requires modification to the apparatus 1 toaccommodate the probe.

Any membrane proteins that insert into a lipid bilayer may be deposited.The membrane proteins may be naturally-occurring proteins and/orartificial proteins. Suitable membrane proteins include, but are notlimited to, (3-barrel membrane proteins, such as toxins, porins andrelatives and autotransporters; membrane channels, such as ion channelsand aquaporins; bacterial rhodopsins; G-protein coupled receptors; andantibodies. Examples of non-constitutive toxins include hemolysin andleukocidin, such as Staphylococcal leukocidin. Examples of porinsinclude anthrax protective antigen, maltoporin, OmpG, OmpA and OmpF.Examples of autotransporters include the NalP and Hia transporters.Examples of ion channels include the NMDA receptor, the potassiumchannel from Streptomyces lividans (KcsA), the bacterialmechanosensitive membrane channel of large conductance (MscLi), thebacterial mechanosensitive membrane channel of small conductance (MscS)and gramicidin. Examples of G-protein coupled receptors include themetabotropic glutamate receptor. The membrane protein can also be theanthrax protective antigen.

The membrane proteins preferably comprise α-hemolysin or a variantthereof. The α-hemolysin pore is formed of seven identical subunits(heptameric). The polynucleotide sequence that encodes one subunit ofα-hemolysin is shown in SEQ ID NO: 1. The full-length amino acidsequence of one subunit of α-hemolysin is shown in SEQ ID NO: 2. Thefirst 26 amino acids of SEQ ID NO: 2 correspond to the signal peptide.The amino acid sequence of one mature subunit of α-hemolysin without thesignal peptide is shown in SEQ ID NO: 3. SEQ ID NO: 3 has a methionineresidue at position 1 instead of the 26 amino acid signal peptide thatis present in SEQ ID NO: 2.

A variant is a heptameric pore in which one or more of the sevensubunits has an amino acid sequence which varies from that of SEQ ID NO:2 or 3 and which retains pore activity. 1, 2, 3, 4, 5, 6 or 7 of thesubunits in a variant α-hemolysin may have an amino acid sequence thatvaries from that of SEQ ID NO: 2 or 3. The seven subunits within avariant pore are typically identical but may be different.

The variant may be a naturally-occurring variant which is expressed byan organism, for instance by a Staphylococcus bacterium. Variants alsoinclude non-naturally occurring variants produced by recombinanttechnology. Over the entire length of the amino acid sequence of SEQ IDNO: 2 or 3, a variant will preferably be at least 50% homologous to thatsequence based on amino acid identity. More preferably, the subunitpolypeptide is at least 80%, at least 90%, at least 95%, at least 98%,at least 99% homologous based on amino acid identity to the amino acidsequence of SEQ ID NO: 2 or 3 over the entire sequence. Amino acidsubstitutions may be made to the amino acid sequence of SEQ ID NO: 2 or3, for example a single amino acid substitution may be made or two ormore substitutions may be made. Conservative substitutions may be made,for example, according to the following table. Amino acids in the sameblock in the second column and preferably in the same line in the thirdcolumn may be substituted for each other:

NON-AROMATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar -charged D E H K R AROMATIC H F W Y

Non-conservative substitutions may also be made at one or more positionswithin SEQ ID NO: 2 or 3, wherein the substituted residue is replacedwith an amino acid of markedly different chemical characteristics and/orphysical size. One example of a non-conservative substitution that maybe made is the replacement of the lysine at position 34 in SEQ ID NO: 2and position 9 in SEQ ID NO: 3 with cysteine (i.e. K34C or K9C). Anotherexample of a non-conservative substitution that may be made is thereplacement of the asparagine residue at position 43 of SEQ ID NO: 2 orposition 18 of SEQ ID NO: 3 with cysteine (i.e. N43C or N17C). Theinclusion of these cysteine residues in SEQ ID NO: 2 or 3 provides thiolattachment points at the relevant positions. Similar changes could bemade at all other positions, and at multiple positions on the samesubunit.

One or more amino acid residues of the amino acid sequence of SEQ ID NO:2 or 3 may alternatively or additionally be deleted. Up to 50% of theresidues may be deleted, either as a contiguous region or multiplesmaller regions distributed throughout the length of the amino acidchain.

Variants can include subunits made of fragments of SEQ ID NO: 2 or 3.Such fragments retain their ability to insert into the lipid bilayer.Fragments may be at least 100, such as 150, 200 or 250, amino acids inlength. Such fragments may be used to produce chimeric pores. A fragmentpreferably comprises the 3-barrel domain of SEQ ID NO: 2 or 3.

Variants include chimeric proteins comprising fragments or portions ofSEQ ID NO: 2 or 3. Chimeric proteins are formed from subunits eachcomprising fragments or portions of SEQ ID NO: 2 or 3. The n-barrel partof chimeric proteins are typically formed by the fragments or portionsof SEQ ID NO: 2 or 3.

One or more amino acid residues may alternatively or additionally beinserted into, or at one or other or both ends of, the amino acidsequence SEQ ID NO: 2 or 3. Insertion of one, two or more additionalamino acids to the C-terminal end of the peptide sequence is less likelyto perturb the structure and/or function of the protein, and theseadditions could be substantial, but preferably peptide sequences of upto 10, 20, 50, 100 or 500 amino acids or more can be used. Additions atthe N-terminal end of the monomer could also be substantial, with one,two or more additional residues added, but more preferably 10, 20, 50,500 or more residues being added. Additional sequences may also be addedto the protein in the trans-membrane region, between amino acid residues119 and 139 of SEQ ID NO: 3. More precisely, additional sequences may beadded between residues 127 and 130 of SEQ ID NO: 3, following removal ofresidues 128 and 129. Additions may be made at the equivalent positionsin SEQ ED NO: 2. A carrier protein may be fused to an amino acidsequence according to the disclosure.

Standard methods in the art may be used to determine homology. Forexample the UWGCG Package provides the BESTFIT program which can be usedto calculate homology, for example used on its default settings(Devereux et al (1984) Nucleic Acids Research 12, p 387-395). The PILEUPand BLAST algorithms can be used to calculate homology or line upsequences (such as identifying equivalent residues or correspondingsequences (typically on their default settings)), for example asdescribed in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. Fet al (1990) J Mol Biol 215:403-10. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nhn.nih.gov/).

The membrane proteins can be labelled with a revealing ordetection/detectable label. The detectable label can be any suitablelabel which allows the proteins to be detected. Suitable labels include,but are not limited to, fluorescent molecules, radioisotopes, e.g. ¹²⁵I,³⁵S, enzymes, antibodies, polynucleotides and linkers such as biotin.

The membrane proteins may be isolated from an organism, such asStaphylococcus aureus, or made synthetically or by recombinant means.For example, the protein may be synthesized by in vitro transcriptiontranslation. The amino acid sequence of the proteins may be modified toinclude non-naturally occurring amino acids or to increase the stabilityof the proteins. When the proteins are produced by synthetic means, suchamino acids may be introduced during production. The proteins may alsobe modified following either synthetic or recombinant production.

The proteins may also be produced using D-amino acids. In such cases theamino acids will be linked in reverse sequence in the C to Norientation. This is conventional in the art for producing suchproteins.

A number of side chain modifications are known in the art and may bemade to the side chains of the membrane proteins. Such modificationsinclude, for example, modifications of amino acids by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄, amidination with methylacetimidate or acylation with aceticanhydride.

Recombinant membrane proteins may be produced using standard methodsknown in the art. Nucleic acid sequences encoding a protein may beisolated and replicated using standard methods in the art. Nucleic acidsequences encoding a protein may be expressed in a bacterial host cellusing standard techniques in the art. The protein may be introduced intoa cell by in situ expression of the polypeptide from a recombinantexpression vector. The expression vector optionally carries an induciblepromoter to control the expression of the polypeptide.

Thus an apparatus of the disclosure, such as apparatus 1 can be used fora wide range of applications. Typically a membrane protein is insertedin the layer 11. An electrical signal, typically a current signal,developed between the electrode 21 in the recess 5 and the furtherelectrode 24 in the chamber 7 is monitored, using the electrical circuit26. Often a voltage is also applied between the electrodes 21 and 24,whilst monitoring the electrical signal. The form of the electricalsignal, and in particular changes therein, provide information about thelayer 11 and any membrane protein inserted therein.

Some non-limitative examples of uses will now described. One use is invitro investigation of membrane proteins by single-channel recording. Animportant commercial use is as a biosensor to detect the presence of arange of substances. The apparatus 1 may be used to detect an analytemolecule that binds with an inserted membrane protein, or anotherstimulus, using stochastic sensing by detecting a change in the currentflow indicating the presence of the analyte molecule or other stimulus.Similarly, the apparatus 1 may be used to detect the presence or absenceof membrane pores or channels in a sample, by detecting a change in thecurrent flow as the pore or channel inserts. The lipid bilayer may beused for a range of other purposes, such as studying the properties ofmolecules known to be present (e.g. DNA sequencing or drug screening),or separating components for a reaction.

Some techniques to reduce or avoid the problem of the pre-treatmentcoating 30 covering the electrode 21 will now be discussed.

A first technique may comprise the following: after application of thepre-treatment coating 30 to apply a voltage across the electrode 21 inthe recess 5 and the further electrode 24 in the chamber 7 sufficient toreduce the amount of excess hydrophobic fluid covering the electrode 21in the recess 5. This is produces a similar effect to electro-wetting.

This technique is illustrated in FIGS. 10A, 10B, 10C, 10D and 10E.First, as shown in FIG. 10A, the pre-treatment coating 30 is applied asshown in FIG. 10A where the pre-treatment coating 30 covers theelectrode 21. Next, as shown in FIG. 10B, aqueous solution 10 is flowedacross the body 2 to cover the recess 5 so that aqueous solution 10flows into the recess 5. Next, a voltage is applied which removes thepre-treatment coating 30 covering the electrode 21, as shown in FIG.10C. This voltage will rupture any layer of amphiphilic molecules formedacross the recess 5. Therefore, next, as shown in FIG. 10D, the aqueoussolution 10 is flowed out of the chamber 7 to uncover the recess 5.Typically an amount of aqueous solution 10 will remain in the recess 5.Lastly, as shown in FIG. 10E, aqueous solution 10, having amphiphilicmolecules added thereto, is flowed across the body 2 to re-cover therecess 5 so that the layer 11 of the amphiphilic molecules forms.

This is most simply performed by flowing the same aqueous solution 10 inand out of the chamber 7. However, in principle, the aqueous solution 10flowed into the chamber 7 to re-covering the recess 5 (in FIG. 10E)could be different from the aqueous solution 10 flowed into the chamber7 to first cover the recess 5 (in FIG. 10B) before applying the voltage.Similarly, there could be no amphiphilic molecules added to the aqueoussolution 10 flowed into the chamber 7 to first cover the recess 5 (inFIG. 10B) before applying the voltage.

A second technique comprises making an inner part of the internalsurface of recess 5 hydrophilic. This may be achieved by making body 2with two (or in general more) further layers 4 a and 4 b as shown inFIG. 11, of which the innermost further layer 4 a (or layers) formed ofa hydrophilic material, for example SiO₂. Typically but withoutlimitation, the innermost further layer 4 a might have a thickness of 2μm.

The outermost further layer 4 b (or layers) is formed of a hydrophobicmaterial and as a result both of (a) the outermost surface of the body 2around the recess and (b) the outer part of the internal surface of therecess 5 extending from the rim of the recess 5 is hydrophobic. Thisassists in the spreading of the pre-treatment coating. In someembodiments, indeed this property of these surfaces of the body 2 isdesirable even if there is not an inner further layer 4 a formed of ahydrophilic material. Typically but without limitation, outermostfurther layer 4 b might have a thickness of 1 μm, 3 μm, 5 μm, 10 μm, 20μm or 30 μm.

A third technique comprises providing a hydrophillic surface onelectrode 21 which repels an applied pre-treatment coating 30, whilstallowing ionic conduction from aqueous solution 10 to electrode 2. Thismay be achieved by depositing a protective material on electrode 21. Arange of protective materials may be used. One possibility is aconductive polymer, for example polypyrrole/polystyrene sulfonate asdiscussed above. Another possibility is a covalently attachedhydrophilic species, such as thiol-PEG.

As will be understood by those skilled in the art who have the benefitof the instant disclosure, other equivalent or alternative devices,methods, and systems for formation of layers of amphiphilic moleculescan be envisioned without departing from the description containedherein. Accordingly, the manner of carrying out the disclosure as shownand described is to be construed as illustrative only. Persons skilledin the art may make various changes in the shape, size, number, and/orarrangement of parts of one or more of the apparatus of the disclosurewithout departing from the scope of the instant disclosure. Similarlyone or more methods of the disclosure may be changed by varying thesteps without departing from the scope of the disclosure. Also, whereranges have been provided, the disclosed endpoints may be treated asexact and/or approximations as desired or demanded by the particularembodiment. In addition, it may be desirable in some embodiments to mixand match range endpoints. A device or parts thereof may be configuredand arranged to be disposable, serviceable, interchangeable, and/orreplaceable. These equivalents and alternatives along with obviouschanges and modifications are intended to be included within the scopeof the present disclosure. Accordingly, the foregoing disclosure isintended to be illustrative, but not limiting, of the scope of thedisclosure as illustrated by the following examples and claims.

Example 1

The apparatus 1 described above has been made and used experimentally todemonstrate formation of a layer 11, in particular being a lipidbilayer, and insertion of a membrane protein, for example α-hemolysin.The following procedure was followed after manufacture of the apparatus1:

1) apply pre-treatment coating 30 to body 2;

2) introduce aqueous solution 10 into chamber 7 to cover recess 5;

3) electro-wet the electrode 21;

4) remove aqueous solution 10 to un-cover recess 5 and re introduceaqueous solution 10 into chamber 7 to cover recess 5 and form the layer11;

5) add α-hemolysin free into aqueous solution 10 and monitor insertioninto layer 11.

In step 1), the pre-treatment coating 30 was hexadecane dissolved inpentane. The quantity and volume of the pre-treatment coating 30 wasvaried for each test to obtain the optimum conditions for formation ofthe layer 11. Insufficient pre-treatment coating 30 prevented formationof the layer 11 while excess pre-treatment coating 30 caused blocking ofthe recesses. However routine variation of the amount allowedoptimisation.

The amphiphilic molecules were a lipid, in particular1,2-diphytanoyl-sn-glycero-3-phosphocholine. The lipid was dissolved inpentane and then dried onto the surface of the cover 6 defining aninternal surface of the chamber 7 before attaching the cover 6 on top ofthe body 2. In step 2), the aqueous solution 10 collected the lipid.

Step 3) was performed by application of a large potential to across theelectrodes 21 and 24. This removed excess pre-treatment coating 30 fromthe electrode 21. Although not required in every case, when performedthis stage helped to condition the recess 5 for formation of the layer11 and assisted subsequent measurement of electrical signals.

By monitoring of the electrical signals developed across the electrodes21 and 24, in steps 4) and 5), formation of the layer 11 and insertionof the membrane protein was observed.

The procedure was successfully performed for an apparatus 1 of the typedescribed above formed by lamination onto a polymer substrate 3.Formation of the layer 11 and insertion of the membrane protein wasobserved using all the fabrication variables described above, albeitwith varying degrees of repeatability and signal quality.

Example 2

An example will now be described for a typical apparatus 1, in which thefirst conductive layer 20 was formed by a silver foil strips (25 μmthick, from Goodfellow) thermally laminated onto the substrate 3 using a15 μm thick laminating film (Magicard) to form the further layer 4. Acircular recess 5 of diameter 100 μm was created further layer 4 usingan excimer laser, exposing a circular silver electrode 21 of diameter100 μm. The exposed silver was chloridised electrochemically asdescribed previously. The second conductive layer 23 was a screenprinting silver/silver chloride ink printed on the top side of the body2.

The pre-treatment coating 30, comprising 0.5 μl of 1% heaxadecane+0.6mg/ml DPhPC in pentane, was then applied to the body 2 and dried at roomtemperature.

The cover 6 comprised a 1 mm thick silicon rubber body with a 250 μmthick Mylar lid. Lipid (4 μl of 10 mg/ml DPhPC in pentane) was appliedto the inside of the cover 6 and allowed to dry at room temperaturebefore attachment to the body 2 with self-adhesive.

A typical successful test proceeded as follows.

The dry contacts 22 and 25 were attached to the electrical circuit 26enclosed in a Faraday cage and a 20 mV 50 Hz triangular potentialwaveform applied. FIG. 15 shows the applied waveform and the resultantcurrent signal which is indicative of the expected capacitive response.

Addition of the aqueous solution 10 creates an “open circuit” connectionbetween the electrodes, such that the current response to the appliedpotential waveform is large, typically saturating the current amplifier.A typical trace is shown in FIG. 16, involving a current responsegreater than 20,000 pA to the 20 mV potential. This corresponds to aresistance of less than 1MΩ, which is sufficiently small for use inconjunction with bilayer formation and pore current measurement.

In the event that the electrode 21 does not initially form a properelectrical connection with the aqueous solution 10, application of a −1VDC potential can be used to increase in the available active electrodearea. This is illustrated in FIG. 17, in which the electrode beginspartially active and is then fully activated after around 4 s of theapplied potential.

Following open-circuit connection between the aqueous solution 10 andthe electrode 21, the aqueous solution 10 is removed from the chamber 7and reintroduced. On re-introduction, a layer 11 of the lipid collectedfrom the internal surface of chamber 7 is formed across recess 5. Theformation is observed by an increase in the capacitive squarewavecurrent response to just under 500 pA, for example as shown in FIG. 18.This value is consistent with the capacitance expected for a circularlipid bilayer of diameter of order 100 μm and varies predictably fordifferent geometries.

Subsequent addition of α-hemolysin to the aqueous solution 10 creates acurrent response typical of pore insertion under an applied potential of100 mV. For example FIG. 19 is a typical example with cyclodextrinpresent in the aqueous solution 10 and shows an expected currentresponse with binding events confirming that the current is through thepores.

Although the example above shows data for the thermally laminatedapparatus 1, the other systems investigated also produced successfulformation of the layer 11 and pore insertion. For example, this was alsosuccessfully demonstrated for an apparatus 1 formed by lamination usingpressure-sensitive adhesive bonding of the further layer 4. However, theadhesive layer was found to complicate formation of the recess 5 both interms of the resulting aspect ratio and spreading of the adhesive acrossthe electrode 21. This problem was overcome by electrical sparking to“activate” the electrode 21.

The impact of the quality of the recess 5 is evident by comparingresults from recesses formed by a CO₂ laser and an excimer laser, asshown in FIGS. 20 and 21, respectively. In both cases formation of thelayer 11 and pore insertion is successful and evident in the response,but more reproducible apertures were produced using the excimer laser.Recesses 5 formed by the CO₂ laser tended to form relatively leakylayers 11 with more noisy pore signals and were also susceptible toblocking. Recesses 5 formed by the excimer laser produced well sealedlayers 11 with good pore signals.

Formation of the layer 11 and pore insertion was similarly observed withan apparatus 1 formed as described above using high definition printedcircuit board manufacture. In this case, to form apparatus 1, the firstconductive layer 20 was formed by etching the copper foil on an FR4substrate typically used in printed circuit board manufacture. The boardwas then screen printed with a Ronascreen SPSR™ photoimageable soldermask to a depth of 25 μm and exposed to UV light on an Orbotech Paragon9000 laser direct imaging machine and developed with KaC0₃ solution tocreate 100 μm circular apertures over the electrodes 21.

Formation of the layer 11 and pore insertion was similarly observed withan apparatus 1 formed as described above using photolithography. In thiscase, to form the apparatus 1, the first conductive layer 20 was formedby gold vapour deposited using clean-room facilities onto the substrate3 and a further layer 4 of SU8 photoresist of thickness 12.5 μm wasspin-coated on top. Recesses 5 were formed by curing of the photoresistby UV exposure with a mask and subsequent removal of the uncuredphotoresist. Recesses 5 had a diameter of 100 μm, exposing an electrode21 of diameter 100 μm. After baking to set the photoresist, the waferwas diced to form separate substrates each with a single recess 5. Theelectrodes 21 were electroplated with silver and then chloridisedelectrochemically as described previously. The second electrode 24 wasscreen printed silver/silver chloride ink printed on the top side of thebody 2.

The pre-treatment coating 30, comprising 0.5 μl of 0.75% hexadecane inpentane, was then applied to the body 2 and dried at room temperature.

The cover 6 comprised a 1 mm thick silicon rubber body with a 250 μmthick Mylar lid. Lipid (4 μl of 10 mg/ml DPhPC in pentane) was appliedto the inside of the cover 6 and allowed to dry at room temperaturebefore attachment to the body 2 with self-adhesive.

Testing was performed as described above and successful formation of thelayer 11 and pore insertion was observed. For example, FIG. 22 shows atypical current trace showing cyclodextrin binding events with wild-typeα-hemolysin pores.

These results generally show the case with which the method of formationof the layer 11 may be performed. In particular formation of the layer11 is achieved with a wide range of materials of the apparatus 1,dimensions (width and depth) of the recess 5, and methods ofmanufacture. Some variation in success rate is evident but in generalthis can be optimised by routine testing of different apparatuses 1. Inparticular the formation of the layer 11 is not overly dependent on thewidth of the recess 5. Formation has been demonstrated over widths fromS 5 m to 100 μm and in view of the ease of formation it is expected thatformation is possible at higher widths up to 200 μm, 500 μm or higher.Also in view of this ease of formation of the layer 11, it is expectedthat variations of the shape of the recess 5 could also be accommodated.

Example 3

There will now be discussed modifications to the apparatus 1 to includeplural recesses 5, commonly referred to as an array of recesses 5. Theability to easily form an array of layers 11 across an array of recesses5 in a single apparatus 1 is a particular advantage of the presentdisclosure. By contrast to traditional methods of formation of lipidbilayers, the apparatus 1 has a single chamber 7, but creates the layer11 in situ during the test and captures a reservoir of electrolyte inthe recess 5 under the layer 11 which allows continuous stablemeasurement of current passing through protein pores inserted in thelayer 11. Further the layer 11 formed is of high quality and islocalised to the area of the recess 5, ideal for high-fidelity currentmeasurements using membrane protein pores. These advantages aremagnified in an apparatus 1 which forms an array of layers 11 becausethis allows measurements to be taken across all the layers 11 inparallel, either combining the current signals to increase sensitivityor monitoring the current signals separately to perform independentmeasurements across each layer 11.

Apparatuses having an array of recesses 5 have been tested anddemonstrated successful formation of an array of layers 11, showing thepossibility of creating a miniaturized array of close packedindividually addressable layers recording current signals in parallelfrom a test sample.

Essentially an apparatus 1 having an array of recesses 5 can be formedsimply using the manufacturing techniques described above but insteadforming plural recesses 5. In this case, the first conductive layer 20is divided to form a separate electrode 21, contact 22 and intermediateconductive track 27 in respect of each recess 5. The apparatus 1 has asingle chamber 7 with a single electrode 24 common to all the recesses5.

FIGS. 23, 24 and 25 show first to third designs in which the apparatus 1is modified by providing, respectively, four, nine and 128 recesses 5 inthe further layer 4. In each of the first to third designs, the firstconductive layer 20 is divided, as shown, respectively, in FIGS. 26, 27and 28 being plan views of the substrate 3. The first conductive layer20 provides, in respect of each recess 5: an electrode 21 underneath therecess 5; a contact 22 exposed for connection of the external circuit 26and a track 27 between the electrode 21 and the contact 22. Thus eachelectrode 21, and its associated track 27 and contact 22, iselectrically insulated from each other allowing separate measurement ofcurrent signals from each recess 5.

Manufacture of the apparatus 1 may be performed using the techniquesdescribed above using lamination of polymer films or photolithographyusing silicon wafers.

Apparatuses 1 having plural recesses 5 have been made and usedexperimentally to demonstrate formation of a layer 11, in particularbeing a lipid bilayer, and insertion of a membrane protein, inparticular α-hemolysin. The experimental procedure was as describedabove for an apparatus 1 having a single recess 5, except that formationof the layer 5 and membrane protein insertion was observed at pluralrecesses 5. Some examples are as follows.

An apparatus of the first design having four recesses 5 was manufacturedby the technique described above of lamination onto a polymer substrate3. The first conductive layer 20 was silver vapour deposited on apolyester sheet substrate 3. The further layer 4 was a 15 μm thicklaminating film thermally laminated on top. The four recesses 5 of 100μm diameter were formed at a pitch of 300 μm by an excimer laser.

For recording of from each recess 5 simultaneously in parallel, multipleAxon current amplifier devices were operated in parallel with a singlesilver/silver chloride electrode 24 in the chamber 7 as the groundelectrode common to all channels. Formation of layers 11 and insertionof membrane proteins at plural recesses 5 was successfully recorded inparallel. Often this occurred at each recess 5 although sometimes alayer 11 failed to form at one or more recesses 5. For example typicalcurrent traces are shown in FIG. 29 demonstrating simultaneous formationof four layers 11, each having one or two α-hemolysin pores inserted,with cyclodextrin binding events. Notably there is no cross-talk betweenthe signals. This confirms that the layers 11 are operatingindependently and can produce meaningful measurements in parallel whilebeing individually addressed and using a common second electrode 24.

An apparatus of the second design having nine recesses 5 wasmanufactured by the technique described above of photolithography usingsilicon wafer substrates 2. The further layer 4 was 5 μm thick SU8photoresist. The nine circular recesses 5 were formed at a pitch of 300μm by photolithography. In this case, the recesses 9 had differentdiameters, in particular of 5 m, 10 μm, 15 μm, 20 μm, 20 μm, 30 μm, 40μm, 50 μm, and 100 μm. The substrate 3 was bonded to a printed circuitboard with separate tracks connected to each contact 22 and 25. Epoxywas added across the contacts 22 and 25 for protection.

In order to control the applied potential and record the currentresponse in parallel, a multichannel electrical circuit 26 was createdwith corresponding software. Testing was computer automated using asyringe pump to provide fluidics control of the repeated application andremoval of the aqueous solution 10.

Formation of layers 11 and insertion of membrane proteins at pluralrecesses 5 was successfully recorded in parallel. Often this occurred ateach recess 5 although sometimes a layer 11 failed to form at one ormore recesses 5. For example, typical current traces for recesses 5constructed with gold electrodes and operating without a redox couple insolution are shown in FIG. 30 demonstrating simultaneous formation ofeight layers 1, each having one or two α-hemolysin pores inserted, withcyclodextrin binding events. Again there is no cross-talk and thisconfirms that the layers 11 are operating independently and can producemeaningful measurements in parallel.

Furthermore the apparatus 1 demonstrates successful formation of a layer11 across the recess 5 of each diameter in the range of 5 μm to 100 μm.Accordingly the apparatus 1 was used to investigate the role of thediameter of the recess 5 and the quantity of pre-treatment coatingapplied, by experimentally testing the percentage success rate offorming a layer 5 with three different concentrations of pre-treatmentcoating 30, namely 0.5%, 1.0%, and 2.0% hexadecane in pentane. Theresults showed that in the case of too little pretreatment coating 30,it was not possible to form the layer 11 across the range of diametersof recess 5. Furthermore in the case of too much pretreatment coating30, it was not possible to wet the electrode 21 and formation of thelayer 11 could not be observed. In this particular configuration, theyield of formation of layers 11 was greater than 60% for the range ofdiameters 15 μm to 100 μm. Factors affecting layer formation, some ofwhich were investigated in this experiment include, but are not limitedto, pretreatment coating 30, diameter of recess 5, depth of recess 5,aspect ratio of recess 5, surface properties of the recess 5, surfaceproperties of the surfaces around the recess, fluid flow within thechamber 7, the amphiphilic molecules used in the layer formation and thephysical and electrical properties of the electrode 21 within the recess5. Subsequent experiments have demonstrated yield of formation of layers11, verified by stochastic binding signals of inserted membranechannels, greater than 70% using the 128 recesses, each 100 μm indiameter, of the device of FIG. 28.

In the apparatus 1 described above, the conductive tracks 27 from theelectrode 21 to the contact 22 is formed on a surface of the substrate 3under the further layer. This may be referred to as a planar escaperoute for the conductive track 27. As previously described the separateconductive tracks 27 allow each electrode 21 to be connectedindividually to a dedicated low-noise high-input impedance picoammeterin the circuit 26 whilst minimizing the signal deterioration due tonoise and bandwidth reduction. Such planar conductive tracks 27 areideal for an apparatus 1 having a small number of recesses 5 and a thicklayer between the tracks 27 and the aqueous solution 10.

However, for uses where high sensitivity is required, the electricalconnection between the electrodes 21 and the amplifier circuit desirablyhas low parasitic capacitance and low leakage to the surroundings.Parasitic capacitance causes noise and hence signal deterioration andbandwidth reduction. Leakage also increases noise, as well asintroducing an offset current. In the apparatus 1, the conductive tracks27 experience some degree of parasitic capacitance and leakage, bothbetween tracks 27 and between track and aqueous solution 10. As thenumber of recesses in the array increases, the number of electricalconnections to escape increases and with a planar escape route, apractical limit is reached where the density of the conductive tracks 27creates too much parasitic capacitance and/or leakage between tracks.Furthermore as the thickness of the layer 4 decreases the capacitanceand/or leakage between the tracks 27 and the aqueous solution 10increases.

By way of example, typical figures may be obtained by modelling thelipid bilayer as a capacitive element with a typical value for thecapacitance per unit area of 0.8 μF/cm². The parasitic capacitancebetween track 27 and aqueous solution 10 can be crudely modelled as acapacitative element with the area of track 27 exposed, through thelayer, to the aqueous solution. Typical values for the track 27 may be50 μm wide with 2 mm exposed and a relative permittivity (dielectricconstant) of the layer around 3. For a 100 μm diameter bilayer and 20 mdeep recess the capacitance is 63 pF with a track-solution parasiticcapacitance of 0.13 pF. However scaling to smaller bilayers of 5 μmdiameter and 1 μm deep the capacitance is 0.16 pF with parasiticcapacitance 0.53 pF. For smaller bilayers and thinner layers theparasitic capacitance dominates.

To reduce this problem, a modification shown in FIG. 31 comprises,replacing the conductive track 27 by a conductive path 28 which extendsthrough the body 2 to a contact 29 on the opposite side of the body 2from the electrode 21. In particular, the conductive path 28 extendsthrough the substrate 3. As this substrate 3 provides a thickerdielectric between the conductive paths 28 than is possible between theplanar conductive paths 27, a much lower parasitic capacitance isachieved. Also, the leakage is low due to the thickness and dielectricproperties of the substrate 3. Consequently, the use of the conductivepaths 28 effectively increases the number of recesses 5 which may beaccommodated in the body 2 before the practical limits imposed byparasitic capacitance and/or leakage are met. This form of interconnectcan be attached to a low-capacitance multi-layer substrate 61, whichallows a far greater number of electrical escape routes by virtue of thenumber of layers and the low dielectric constant of the material. Inaddition the use of solder bump technology (also known as “flip chip”technology) and a suitable connector allows the apparatus 1 shown inFIG. 31, excluding the substrate 61, to be made as low cost disposablepart.

The conductive path 28 may be formed using known through-waferinterconnection technology. Types of through-wafer interconnects whichmay be applied to form the conductive path include without limitation:

on substrates 3 of silicon, through-wafer interconnects formed byproducing a via through the silicon wafer, isolating the internalsurface of via and filling the via with a conducting material, oralternatively the conductive path 28 is formed by producing asemiconductor PN junction in the form of a cylindrical via through thesilicon substrate; on substrates 3 of glass, through-wafer interconnectsformed by methods including laser drilling, wet etching and filling viaswith metal or doped semiconductor material; and on substrates 3 made ofpolymers, through-wafer interconnects formed by methods including laserdrilling, laser ablation, screen printed conductors and known printedcircuit board techniques.

As the opposite side of the body 2 from the electrode 21 is dry, anelectrical point contact array can be used to make connections to theelectrical circuit 26. By way of example, FIG. 31 illustrates the use ofsolder bump connections. In particular, deposited on each contact 29 arerespective solder bumps 60 on which a circuit element 61 is mounted sothat the solder bumps 60 make electrical contact with a track 62 on thecircuit element 61.

The circuit element 61 may be a printed circuit board for example asshown in FIG. 13.

Alternatively, the circuit element 61 could be an integrated circuitchip or a laminate, for example a low temperature cured ceramic package.Such an integrated circuit chip or laminate may be used as a method ofspreading out connections, connecting to a further solder bump array onthe opposite side of the integrated circuit chip or laminate with agreater pitch. An example of this is shown in FIG. 32 in which thecircuit element 61 is an integrated circuit chip or a laminate providingconnections from the solder bumps 60 deposited on the body 2 to furthersolder bumps 63 arrayed at a greater pitch and used to connect to afurther circuit element 64, for example a printed circuit board. Thecircuit element 61 being an integrated circuit chip or laminate may alsobe used to escape connections sideways in a multi-layer format.

In the case of a substrate 3 of semiconductor material such as silicon,two types of through-wafer interconnect which may be applied to make theconductive path 28 are Metal-Insulator-Semiconductor (MIS), and a PNjunction type. In MIS, a hole is drilled through the silicon chip byDeep Reactive Ion Etching (DRIE) process and this hole is coated withinsulator and then filled with metal to forma the conductive path 28.The PN junction type of through-wafer interconnect is a semiconductorjunction formed into a cylindrical via through a silicon chip. Each typeof through-wafer interconnection is formed on silicon wafers that havebeen thinned down to less than 0.3 mm to save DRIE processing time inmaking the holes. The important feature of PN junction typethrough-wafer interconnects is the low capacitance provided by having alarge depletion region compared to the MIS type of interconnect. This ispartially helped by increasing the reverse-bias of the junction.

The invention claimed is:
 1. An apparatus for supporting a layerseparating two volumes of aqueous solution, the apparatus comprising:elements defining a chamber, the elements including a body ofnon-conductive material having formed therein at least one recessopening into the chamber; and an electrode contained in the recess;wherein an outer part of an internal surface of the recess ishydrophobic; wherein an inner part of the internal surface of therecess, closer to the electrode than the outer part of the internalsurface of the recess, is hydrophilic; and wherein the body comprises anoutermost layer formed of a hydrophobic material and an inner layerformed of a hydrophilic material, the recess extending through both theoutermost layer and the inner layer, said outer part of the internalsurface of the recess being a surface of the outermost layer, and saidinner part of the internal surface of the recess being a surface of theinner layer.
 2. An apparatus according to claim 1, wherein surfacesincluding either or both of (a) an outermost surface of the body aroundthe recess, and (b) at least an outer part of an internal surface of therecess extending from the rim of the recess, are hydrophobic.
 3. Anapparatus according to claim 2, wherein said outer part of the internalsurface of the recess is a surface of the outermost layer.
 4. Anapparatus according to claim 2, wherein said surfaces are modified by afluorine species.
 5. An apparatus according to claim 4, wherein saidsurfaces are modified by a fluorine species by treatment with a fluorineplasma.
 6. An apparatus according to claim 1, wherein the electrodecontained in the recess is provided on the base of the recess.
 7. Anapparatus according to claim 1, wherein the body comprises a substrateand at least one further layer attached to the substrate, the recessextending through the at least one further layer.
 8. An apparatusaccording to claim 7, wherein the at least one further layer is:polycarbonate; poly-vinyl chloride; polyester; a thermal laminatingfilm; a photoresist; or an ink.
 9. An apparatus according to claim 7,wherein the substrate comprises at least one of silicon, silicon oxide,silicon nitride or a polymer.
 10. An apparatus according to claim 7,wherein the body has a conductive path extending from the electrode to acontact allowing connection to an electrical circuit.
 11. An apparatusaccording to claim 10, wherein the conductive path extends through thebody to a contact disposed on the opposite side of the body from therecess.
 12. An apparatus according to claim 10, wherein the conductivepath extends across a surface of the substrate under the at least onefurther layer.
 13. An apparatus according to claim 1, wherein theelectrode has provided thereon a hydrophilic surface which repels ahydrophobic fluid whilst allowing ionic conduction from an aqueoussolution to the electrode.
 14. An apparatus according to claim 13,wherein the hydrophilic surface is a surface of a protective materialprovided on the electrode.
 15. An apparatus according to claim 14,wherein the protective material is a covalently-attached hydrophilicspecies or a conductive polymer.
 16. An apparatus according to claim 1,wherein the electrode has a conductive polymer provided thereon.
 17. Anapparatus according to claim 1, further comprising a further electrodein the chamber outside said recess.
 18. An apparatus according to claim1, wherein the elements defining the chamber further include a coverextending over the body so that the chamber is a closed chamber.
 19. Anapparatus according to claim 18, wherein the cover comprises at leastone inlet and at least one outlet, wherein the inlet is configured forthe introduction of an aqueous solution into the chamber and the outletis configured to vent fluid displaced by any aqueous solution thusintroduced.
 20. An apparatus according to claim 1, wherein the internalsurface of the recess has no openings capable of fluid communication.21. An apparatus according to claim 1, wherein the recess has a width ofat most 500 μm.
 22. An apparatus according to claim 1, wherein the atleast one recess is plural recesses.
 23. An apparatus according to claim1, further comprising amphiphilic molecules deposited on an internalsurface of the chamber.
 24. An apparatus according to claim 23, whereinthe amphiphilic molecules are lipids.
 25. An apparatus according toclaim 1, further comprising a membrane protein deposited on an internalsurface of the chamber.
 26. An apparatus according to claim 1, furthercomprising a pre-treatment coating of a hydrophobic fluid on a surfaceof the body around the recess and/or on an internal surface of therecess.
 27. An apparatus according to claim 26, wherein the recess andthe chamber contain aqueous solution.
 28. An apparatus according toclaim 27, further comprising a layer of amphiphilic molecules extendingacross the opening of the recess.
 29. An apparatus according to claim28, wherein the layer of the amphiphilic molecules has an electricalresistance of at least 1GΩ.
 30. An apparatus according to claim 28,wherein the amphiphilic molecules are lipids.
 31. An apparatus accordingto claim 28, the layer of amphiphilic molecules having a membraneprotein inserted therein.